17th world congress of the international society on

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Toxicon 60 (2012) 95–248

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Toxicon

journal homepage: www.elsevier .com/locate/ toxicon

17th World Congress of the International Society on Toxinology&

Venom Week 2012Honolulu, Hawaii, USA, July 8–13, 2012

Abstract Editors:Steven A. Seifert, MD and Carl-Wilhelm Vogel, MD, PhD

0041-0101/$ – see front matter10.1016/j.toxicon.2012.04.354

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http://dx.doi.org/10.1016/j.toxicon.2012.04.354

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Abstracts Toxins 2012 / Toxicon 60 (2012) 95–24896

Abstracts Toxins 2012

Contents

A. Biotoxins as Bioweapons. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 97B. Drug Discovery & Development. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 100C. Evolution of Toxins & Venom Glands . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 119D. Hemostasis. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 128E. History of Toxinology . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 137F. Insects . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 141G. Marine . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 144H. Microbial Toxins. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 157I. Miscellaneous . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 164J. Pharmacology . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 165K. Physiology . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 175L. Plants . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 177M. Scorpions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 180N. Snakes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 191O. Spiders. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 228P. Systems & Training . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 236Q. Venomous Animal Biology . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 239R. Venomous Animal Collections . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 242S. Veterinary Toxinology . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 246

This issue of Toxicon contains the abstracts presented at the17th World Congress of the International Society on Tox-inology (IST), held jointly with Venom Week 2012, 4thInternational Scientific SymposiumonAll Things Venomous,in Honolulu, Hawaii, USA, July 8–13, 2012.

The World Congress of IST is held every three years,most recently in Recife, Brazil in March 2009. The ISTWorld Congress is the primary international meetingbringing together scientists and physicians from aroundthe world to discuss the most recent advances in thestructure and function of natural toxins occurring invenomous animals, plants, or microorganisms, in medical,public health, and policy approaches to prevent or treatenvenomations, and in the development of new toxin-derived drugs. The Venom Week Symposiums, previouslyheld in 2005, 2007 and 2009, are held in the USA with aninternational faculty and focus, and present the mostrecent advances in terrestrial and marine envenomationsof medical and veterinary importance, in venom andantivenom basic science, in the handling and breeding ofvenomous animals in zoos and aquaria, in venomousanimal science, and in regulatory issues. As both meetingswere scheduled to be held in the summer of 2012 in theUSA, the two organizations decided to hold a jointmeeting in Honolulu.

The program of the meeting is characterized by twoparallel tracks, basic sciences and clinical sciences, therebyproviding venues for presenting advances in these twobroad areas of toxinology research as well as translationalaspects that bridge these two components. Some sessionson important topics are organized by eminent scientistsin their field, whereas other sessions were developedfrom the more than 300 abstracts submitted to themeeting.

The joint meeting of the 17th World Congress of the ISTand Venom Week 2012 is intended to be a forum ofscientific exchange among the members of the toxinologycommunities from around the globe. It is hoped that theunique location of Hawaii enhances the meeting experi-ence. Hawaii is indeed a very special place. The mostnorthern part of Polynesia, it is the most isolated land massin theworldwith a sizable population. It is characterized byan unparalleled cultural diversity, where the East meets theWest, and where the host culture is Hawaiian. Althoughpart of the United States, it is a fitting place to serve as hostfor the Asia-Pacific Region of the IST for its World Congress.Oahu, the island on which Honolulu is located, isa Hawaiian name and means “Gathering Place”. Appropri-ately, it will be the gathering place for the world toxinologycommunity in 2012.

Many thanks go to the Executive Committee of IST, Pres-ident Dr. Ponnampalam Gopalakrishnakone (Singapore),Secretary/Treasurer Dr. Julian White (Australia), andPresident-Elect Dr. Alan Harvey (United Kingdom), to themembers of both the international Scientific OrganizingCommittee, the Local Organizing Committee, and to themany sponsors for helping to make the 17th IST WorldCongress / VenomWeek 2012 Meeting a reality and success.

Meeting Co-ChairsSteven A. Seifert, MD (USA)Carl-Wilhelm Vogel, MD, PhD (USA)Members of the Scientific Organizing CommitteeAlejandro Alagon (MEXICO)Greta Binford (USA)Leslie V. Boyer (USA)Sean P. Bush (USA)Juan J. Calvete (SPAIN)Jean-Philippe Chippaux (BENIN)

Abstracts Toxins 2012 / Toxicon 60 (2012) 95–248 97

Yara Cury (BRAZIL)Richard C. Dart (USA)P Gopalakrishnakone (SINGAPORE)Eugene Grishin (RUSSIA)Michael Gurevitz (ISRAEL)José María Gutiérrez (COSTA RICA)Abdulrazaq G. Habib (NIGERIA)Alan Harvey (UK)Dan E. Keyler (USA)Glenn King (AUSTRALIA)Jessi Krebs (USA)Stephen P. Mackessy (USA)Ashis k. Mukherjee (INDIA)Kini R Manjunatha (SINGAPORE)Cesare Montecucco (ITALY)Dietrich Mebs (GERMANY)Tarek R. Rahmy (EGYPT)Karen Seibold (USA)Steven A. Seifert (USA)Denis Servent (FRANCE)Reto Stöcklin (SWITZERLAND)Denise V. Tambourgi (BRAZIL)Toru Tamiya (JAPAN)Jan Tytgat (BELGIUM)Carl-Wilhelm Vogel (USA)Julian White (AUSTRALIA)

Members of the Local Organizing CommitteeBrian E. Hew (Black Ivory Biotech, Aiea, Hawaii)Ben Okimoto (Honolulu Zoo, Hawaii)John M. Pezzuto (University of Hawaii at Hilo)Andrew Rossiter (Waikiki Aquarium, Hawaii)Steven A. Seifert (University of New Mexico)Carl-Wilhelm Vogel (University of Hawaii at Manoa)JulianWhite (Women’s andChildren’s Hospital, Adelaide)Catherine Wood (University of New Mexico)

SponsorsRare Disease Therapeutics, Inc., Franklin, Tennessee, USABTG International, Ltd., London, UKInstituto Bioclon, Mexico City, MexicoSilanes Laboratories, S.A., MexicoToxicon American International Rattlesnake Museum,Albuquerque, NM, USA Chiricahua Desert Museum,Rodeo, NM / Elsevier B.V., Amsterdam, The NetherlandsInternational Society on ToxinologyCollege of Pharmacy, University of Hawaii at Hilo,Honolulu, HI University of New Mexico Health SciencesCenter, Albuquerque, NM, USAMidwest Tongs, Greenwood, MO, USA

Meeting OrganizersCarl-Wilhelm Vogel, MD, PhDUniversity of Hawaii Cancer Center, and Department ofPathology, John A Burns School of Medicine, Universityof Hawaii at Manoa, 1236 Lauhala Street, Honolulu, HI96813 USAEmail: [email protected]

Steven A. Seifert, MDDepartment of Emergency Medicine, University of NewMexico School of Medicine, and New Mexico Poison and

Drug Information Center, MSC09 5080, 1 University of NewMexico, Albuquerque, NM 87131-0001 USAEmail: [email protected]

A. Biotoxins as Bioweapons

1. Detection Technologies for Biological Toxins

P. GopalakrishnakoneVenom &Toxin Research Programme, YLL School of Medicine, NationalUniversity of Singapore, SingaporeE-mail address: [email protected].

Review: The ad hoc group of the states parties to “theconvention on the prohibition of the development, produc-tion and stock piling of biological and toxin weapons and ontheir destruction” has listed the following toxins, bacter-iotoxins (Botulinum toxin, clostridium perfringens toxins,staphylococcal enterotoxins, shigatoxins), Phycotoxins (Ana-toxins, ciguatoxins, saxitoxins), Mycotoxins (trichothecenes),Phytotoxins (Abrins,Ricins) Zootoxins (Bungarotoxins). Inaddition, few other toxins which are not in this list such asbrevetoxins, conotoxins and other neurotoxins also will bereviewed in addition to the work done by venom and toxinresearch programme at National University of Singapore.

Anoverviewof thedifferent technologies available for thedetection of biological toxins, originating from animals,plants and microbial organism will be described. The meth-odologies/technologies vary from fieldable ones to highlysensitive one to user friendly ones. Depending on thepurpose of detection of toxin vary in different circumstance,quantitatively or qualitatively starting from Ouchterlonydiffusion test to classical ELISA kit which has been used formany years for the detection of biological toxins as well asHPLC, LCMS. In the recent past our research group hasdeveloped immunosensitive field – Effect Transistors (ISFET)(1), ultrasensitive protein array based on electrochemicalenzyme immunoassay (EETA) (2), silicon chip base opticalimmunoassay (OIA) (3) for the detection of toxins fromanimal venoms as well as toxin from microbial organismssuch as Botulinum toxins, SEB toxin, T-2 toxins as well asplant toxins such as ricin or abrin.

References

Zakaria.E.S, Gopalakrishnakone, P. & Pavel, N. Immunolog-ically sensitive field effect transistors. Encyclopedia ofMedical Devices and Instrumentation. Ed: Weber JC, Pubs:John Wiley & Sons, 2006, p. 98-110.Le, V.D., Fang, X., ZichaoY., Gopalakrishnakone, P. &Gao, Z. Anultrasensitive protein array based on electrochemicalenzyme immunoassay. Frontiers in Biosciences 10, 1654-1660, 2008.Saravanan, G.,Rajasekar, G. & Gopalakrishnakone,P. Fieldable Optical Immunoassay for toxin detection.Ler, S.G, Lee, F.K. & Gopalakrishnakone, P. Trends in detectionof warfare agents: Detection methods for Ricin, SEB and T-2toxin. Journal of Chromatography A,1133,1-12(2006).

Keywords: biological toxins, detection, lethal10.1016/j.toxicon.2012.04.002

mailto:[email protected]




2. Medical Aspects of Bioterrorism

Mahdi Balali-MoodMashhad University of Medical Sciences, School of Medicine, MedicalToxicology Research Center, Mashhad, IranE-mail address: [email protected].

Introduction: Bioterrorism is a terrorist actioninvolving the intentional release or dissemination ofa biological warfare agent (BWA), which includes somebacteria, viruses, rickettsiae, fungi or toxins. BWA isa naturally occurring or human-modified form that maykill or incapacitate humans, animals or plants as an act ofwar or terrorism. BWA is a weapon of choice for massdestruction and terrorism, because of there is usually anincubation period, lesser quantities are required thanchemical warfare agents, they are easily distributed withdevices that are readily obtained, they are usually odor-less, colorless, difficult to detect, with no need ofspecialized equipment for production. BWA may bedisseminated as an aerosol, spray, explosive device, and byfood or water.

Classification: Based on the risk, BWAs have been prior-itized into three categories of A, B and C. Category A includesmicroorganisms or toxins that easily spread, leading tointoxicationwith high death rates such as Anthrax, Botulism,Plague, Smallpox, Tularemia and Viral hemorrhagic fevers.Category B has lower toxicity with wider range, includingStaphylococcal Entrotoxin type B (SEB), Epsilon toxin ofClostridium perfringens, Ricin, Saxotoxins, Abrin andTrichothecenemycotoxins. TheC category includes emergingpathogens that could also be engineered for mass spreadsuch as Hanta viruses, multidrug-resistant tuberculosis,Nipah virus, the tick-borne encephalitis viruses, hemorrhagicfever viruses and yellow fever.

Clinical manifestations of biotoxins in human: Clin-ical features and severity of intoxication depend on theagent and exposed dose, route of entry, individual variationand environmental factors. Onset of symptoms varies from2-24 hours in Ricin to 24-96 hours in Botulism. Clinicalmanifestations also vary from irritation of the eyes, skinand mucus membranes in T2 toxin to an acute flaccidparalysis of bilateral cranial nerve impairment ofdescending manner in botulism. Most of the pyrogenictoxins such as SEB produce the same signs and symptomsas toxic shock syndrome including a rapid drop in bloodpressure, elevated temperature, and multiple organ failure.

Management: There is no specific antidote or effectivetreatment for most of the biotoxins. The clinical manage-ment is thus more supportive and symptomatic. Fortu-nately vaccines are now available for most of BWA.Therefore, immunization of personnel at risk of exposure isrecommended.

Conclusion: Biotoxins are very wide and bioterrorism isa heath and security threat that may induce national andinternational problems. Therefore, health professionals andthe general population should be aware of the potential forbioterrorism.

Keywords: biotoxin, bioterrorism, biological warfare agent10.1016/j.toxicon.2012.04.003

3. Capsaicin: A Novel Antidote against BotulinumNeurotoxin A.

Baskaran Thyagarajan, Shannon Schreiner,Padmamalini BaskaranUniversity of Wyoming School of Pharmacy, College of Health Sciences,Laramie, WY, USAE-mail address: [email protected] (B. Thyagarajan).

Review: Botulinum neurotoxin A (BoNT/A) is the mostpotent toxin produced by Clostridium botulinum. BoNT/Aholotoxin consists of a 100 kDa heavy chain (Hc) and a 50kDa light chain (Lc) linked by a single disulfide bond. BoNT/AHc binding to its receptors on the neural membrane sets thestage for endocytosis into the Motor Nerve Terminals(MNT). Within early endosomes, BoNT/A undergoes pHdependent separation of the Hc and Lc. The Hc translocatesthe Lc into the cytosol where it cleaves SNAP-25 andinhibits neurotransmission. The ease of production andtransportation accounts for the accidental or intentionalmisuse of BoNT/A. The extensive use of BoNT/A to treatclinical diseases also presents opportunities for suchmisuse.BoNT/A, as a bioweapon, can be disseminated via aerosol orby contamination of water or food supplies, to cause wide-spread casualties. The need for prolonged intensive care andventilatory support for the affected patients will imposesevere economic burden on our health care system. Untilnow, no therapeutic strategies are available to treat BoNT/Aintoxication. The antitoxins that are available will be of nouse once BoNT/A has entered the MNT. Therefore, devel-oping therapeutic drug strategies to treat botulism is ofprimary importance. Our research work demonstratesa novel anti-BoNT/A effect of capsaicin, an active ingredientof chili peppers. Capsaicin, a transient receptor potentialvanilloid receptor 1 agonist, demonstrated a prophylactic aswell as a therapeutic effect against BoNT/A. Preinjection ofcapsaicin at the mouse hindlimbs protected the uptake andneuroparalytic effects of BoNT/A (Thyagarajan et al. 2009).The prophylactic effects of capsaicin were abrogated bycapsazepine, a TRPV1 antagonist. Injection of capsaicin, at 12and 24 hr post BoNT/A, remarkably accelerated the recoveryof animals from BoNT/A mediated neuroparalysis. In vivo,capsaicin injected mice showed better performance onrotarod, demonstrated higher compound muscle actionpotential and muscle strength. Also, in vitro, capsaicintreatment potentiated the restorative effects of 2,4 dia-minopyridine (DAP; a potassium channel blocker). Capsaicininjection enhanced recovery by shortening the duration ofneuroparalysis by 50% compared to BoNT/A alone (nocapsaicin) injected controls.

Conclusions: Collectively, we demonstrate that capsa-icin has the potential to be developed as a novel agent toprevent, and to treat BoNT/A intoxication. This discoverysignificantly advances our current knowledge of BoNT/Aantagonists and forms the basis of novel therapeuticapproach from the natural product, chili peppers. Furtherresearch to understand the mechanisms of action ofcapsaicin is in progress.

Keywords: capsaicin, Botulinum neurotoxin A, neuromuscular junction10.1016/j.toxicon.2012.04.004




4. Biohazards of Botulinum Neurotoxins

Ornella RossettoDepartment of Biomedical Sciences, University of Padova, ItalyE-mail address: [email protected].

Review: Botulinum neurotoxins (BoNTs) are produced bytoxigenic strains of C. botulinum in seven serotypes (namedAthrough G). They are responsible for the clinical syndrome ofbotulism blocking the acetylcholine release from peripheralnerves and thus causing a flaccid paralysis. Respiratoryfailure secondary to paralysis of the respiratory muscles canlead to death unless appropriate therapy is promptly initi-ated. Structurally, BoNTs are composed of three domains:a Light Chain (LC), which acts in the cytosol as a metal-loprotease and cleaves protein components (SNAREs) of theneuroexocytosis apparatus; a translocation domain (HN) anda receptor binding domain (HC). The high affinity binding,due to a double receptor interaction and the extremelyspecific enzymatic activity, make botulinum toxin the mostlethal substance known, with a LD50 of 1 nanogram of toxinper kilogram body mass. Nevertheless, it also serves asa remarkably effective treatment for hypercholinergicdisorders such as blepharospasm, strabismus, hemifacialspasm, certain types of spasticity and other ailments and forcosmetic anti-aging treatments. Due to the severity andpotency of this neurotoxin, its importance as a biologicalweapon is of major concern to public health officials and, infact, it belongs to “category A” of agents which representserious risk when misused for military and/or criminalactivities. Despite its widespread accessibility and highlethality, however, BoNThas never been used successfully forpurposes of warfare or bioterrorrism. Several countriesinvestigated botulinum toxin as a biologic agent sinceWorldWar II. Botulism's sole link to a presumed successful planneduse as a weapon is provided by Paul Fildes, a high-rankingBritish specialist in bacterial weapons development duringWorldWar II, who has alluded to the fact that he contributedto the assassination of Reinhard Heydrick, head of theGestapo.More recently, in the early 1990s, before their attackwith Sarin on the Tokyo subway system, the Japanese cultAum Shinrikyo, had released an ineffectively produced C.botulinum preparation in Japan. During the United Nations’inspections of Iraq's capabilities for biologic warfare in 1991,botulinum toxin was clearly an area in which research hadbeen directed.More botulinum toxinwas produced than anyother weaponizable agent in Iraq. Botulinum toxin can beabsorbed by either gastrointestinal or respiratory epitheliumbut does not penetrate intact skin. The clinical forms ofbotulismdependon themodeof contamination but botulismthrough inhalation can only be the result of a deliberate actusing an aerosol.

Keywords: botulinum toxins, botulism, bioterrorism10.1016/j.toxicon.2012.04.005

5. Bioterrorism and Biological Toxins

Cesare MontecuccoDepartment of Biomedical Sciences, University of Padova, ItalyE-mail address: [email protected].

Review: The use of biological agents as a warfare goesback to ancient time, but it is only in modern times thatseveral countries have developed programs of research anddevelopment of biological warfare. More recently, initia-tives to control the possible use of these weapons haveemerged and three lists (A, B and C) of biological agents ofdifferent potential danger have been drawn. In thispresentation I will discuss one toxin producing bacteriumincluded in list A: Bacillus anthracis and two plant proteintoxins included in list B. The mechanisms of action of thetwo anthrax toxins and their immunosuppressive proper-ties will be illustrated together with the mechanism ofaction and pathogenetic consequences of the poisoningwith ricin or abrin will be illustrated. Finally, possiblemethods of preventing their action will be considered.

Keywords: anthrax toxin, ricin, abrin, bioterrorism10.1016/j.toxicon.2012.04.006

6. The Diverse Roles of Botulinum Toxins

Jeffrey BrentToxicology Associates, University of Colorado School of Medicine, Denver,Colorado, USAE-mail address: [email protected].

Review: Botulinumneurotoxin is a cause of the naturallyoccurring disease botulism, a pharmaceutical, and a poten-tial biowarfare/terrorism agent. The bacterium Clostridiumbotulinum is the primary source of this toxin in humandisease. Clostridium botulinum exists as serotypes A-G,although human disease is almost entirely restricted totypes A, B, and E. Up to now there has been no systematicassessment of the differences in disease presentation bydifferent strains. Because botulinum neurotoxins are themost potent substances known, the CDC has listed it asa type A biothreat. The neurotoxin consists of a 150 KDamolecule comprised of a 100 kDA heavy chain (HC) and a 50kDA light chain (LC). Its toxicity results from its interactionwith cholinergic nerve terminals at the neuromuscularjunction (NMJ). TheHC is responsible for facilitating the pH-dependent endocytotic translocation of the toxin intomotor nerve terminals at the NMJ. Once internalized the LCcleaves the SNARE proteins that are responsible for facili-tating the release of acetylcholine into the NMJ. Theresulting failure of acetylcholine release is responsible forthe subsequent loss of skeletal muscle function and theensuing paralytic syndrome. However, the various strains ofbotulinum toxin do not all cleave the same SNARE proteins.The degree to which these differences influence the clinicalpresentation has not been well characterized. There arethree potential preventive and therapeutic approaches tobotulism syndromes: vaccines, pharmacologic interven-tions and therapeutic antibodies. The immunologicmeasures almost certainly are only effective before inter-nalization of the neurotoxin. Vaccines, therefore, areunlikely to be effective therapeutically. Vaccines, however,have been used for many years to protect researchers ormilitary personnel who are to be at risk for encounteringbotulinum toxin. Such vaccinations are effective in inducingneutralizing antibodies. Therapeutic antibodies directed





against botulinum toxin represent the most promising ofthe post-exposure therapeutic approaches on the short-term horizon. They must be given before a clinicallysignificant amount of neurotoxin is internalized into theNMJ. In the US there is currently one approved therapeuticantibody preparation. A number of pharmacologic agentsare currently being studied, however none have yet showngreat promise. Theoretically these might be effective in thepost-NMJ internalization phase. Currently we are assessingthe diversity in the presentations of botulinum syndromescaused by various strains. It is anticipated that elucidationof these difference might provide information on potentialtherapeutic approaches to botulism syndromes. Results ofthese studies will be presented.

Keywords: Botulism, neurotoxin, biothreat10.1016/j.toxicon.2012.04.007

7. Biological Weapons and Toxins: A Short History andLook at the Future

Barbara B. PriceASA, Inc. and International Institute of NonProliferation Studies, IINPS,Kaneohe, HI, USAE-mail address: [email protected].

Review: This review is a look back at the past and intothe future; of the history of toxins used as weapons, and thepossible dangers facing us in the future. Before 2001, therewas recognition that biological weapons, including toxins,were weapons that required state sponsorship. But that isno longer true. As the asymmetries of weapons and politicshave changed, so has the probable use of toxins asweapons. Our understanding and expectation of likely useof toxins in weapons has changed. Even the word “toxin” iscommonly used inappropriately to mean toxic chemicalrather than a chemical produced by organisms. Prior to2001, toxins were evaluated on their ability to be manu-factured or purified from natural resources. Many infec-tious disease specialists regard bacterial toxins as theeventual cause of cell damage and cell death. Where are wein the spectrum of toxins as weapons, from a clear use ofbiological weapons to a purified chemical? Do we needpurified chemicals? Are toxins the ultimate dual useagents? Do toxinologists need to examine their publica-tions for possible illicit use?

Keywords: toxin, biological weapon, bioterrorism10.1016/j.toxicon.2012.04.008

8. Biotoxins and Bioterrorism: Ricin and Saxitoxin

Peter G. BlainMedical Toxicology Centre, Faculty of Medical Sciences, Newcastle University,Newcastle upon Tyne, UKE-mail address: [email protected].

Introduction:Natural biotoxins have been considered bynation states for use as chemobiological weapons, andseveral specifically developed for weaponisation. Ricingained notoriety when it was allegedly used as an agent of

assassination, and saxitoxin was the key component ina weapon system designed to target individuals and beunattributable. Terrorist and extremist groups have includedbiotoxins in their planning, and documents describing theextraction and preparation of ricin have been found in thepossession of terrorist cells.

Toxicology: Ricin is a glycoprotein found in the seedsof the castor oil plant (Ricinus communis). The nativemolecule consists of two polypeptide chains, A and B,which are linked by a disulphide bond that is easilyreduced. On systemic absorption, the B chain (a lectin)binds to cell surface receptors present on eukaryote cellsbut not in prokaryotes. There is endocytic uptake of thetoxin into the cytosol where the chains dissociate and theA chain binds to the 28s ribosomal subunit, blockingprotein synthesis and resulting in the death of the cell.Clinical features develop over several hours and, in onenotorious case, death occurred 3 days after injection ofa pellet containing ricin. Saxitoxins are a group ofcompounds produced by a variety of marine dinoflagel-lates and freshwater cyanophytes (blue-green algae).Ingestion of shellfish that have extracted and concen-trated these toxins may result in paralytic shellfishpoisoning in humans. This toxic effect results from highaffinity binding of saxitoxin to voltage dependent sodiumchannels in the membranes of excitable cells that theninhibits depolarization. Peripheral nerves appear to beparticularly susceptible.

Medical Countermeasures: Symptomatic and supportivetreatments are the primary approach to the medicalmanagement of patients poisoned with either of these bio-toxins. A ricin anti-toxin is under development and assess-ment, but currently there is no antidote to saxitoxinpoisoning.

Conclusion: The biotoxins ricin and saxitoxin are rela-tively easily weaponised and highly toxic to humans. Assuch they are bioterrorist threat agents, especially sincestate programmes have demonstrated their potential use inassassination.

Key words: biotoxins, bioterrorism, ricin, saxitoxin10.1016/j.toxicon.2012.04.009

B. Drug Discovery & Development

9. Genotoxic Potential of Micrurus corallinus Venom onthe DNA of Human Lymphocytes

Silvana Marcussi, C. Trento Marcus Vinicius,Mateus W. EleutérioDepartment of Chemistry, Federal University of Lavras - UFLA, Lavras, MG,BrazilE-mail address: [email protected] (C.T. Marcus Vinicius).

Background: The articles that report the effects ofvenoms and their toxins, isolated from Brazilianwildlife, onhuman DNA are restricted to Apis mellifera and Crotalus d.terrificus. There is little information on the genotoxic and/ormutagenic potential of venoms and isolated toxins of broadmedical and scientific interest, but due to the toxic and





pharmacological effects induced by them, the studies thatcharacterize their action becomes important. Thus, theobjective of this study was to investigate the effects of suchvenom on the DNA of human lymphocytes, in vitro, usingboth the comet assay and cytokinesis-blocked micronu-cleus test.

Methods: Treatments were performed on whole bloodat different venom concentrations; upon testing comple-tion, nucleoids and binucleated lymphocyte cells have beenanalyzed by comet assay and micronucleus test, respec-tively. The cells were maintained for 3 hours in the pres-ence of venom samples for the comet assay, and for72 hours, during cultivation, in the presence of a cytoki-nesis-blocking agent (cytochalasin B) for the micronucleustest. The comet assay and micronucleus test were per-formed by means of fluorescence microscope and opticalmicroscope, respectively.

Results: We were able to observe different levels ofdamage (<5%, 5-20%, 20-40%, 40-85% and >85%) for thecomet assay, except at concentration of 10 mg/mL, which didnot induce damage above 85%. It seems that this venom hasa dose-dependent genotoxic response, since higher concen-trations resulted in increased genotoxic potential. However,all treatments induced damage lower than that observedwhen doxorubicin (drug control) was used. The presence ofmicronuclei in binucleated cells indicates the transmission ofDNA damage to the first generation of treated cells, thuscharacterizing a genotoxic potential. Only concentrations of15 and 30mg/mLwere able to induce the formation of 3.6 and4.1 micronuclei/1000 binucleated cells, respectively, valueshigher than those observed in negative control, 1.4, andlower than the positive control, 12.8 (cisplatin 6 mg/mL).

Discussion: The results of the comet assay and micro-nucleus test complement and confirm the genotoxicpotential of the M. corallinus venom, with emphasis onconcentrations equal to and higher than 15mg/mL. Similareffects were observed for the C. d. terrificus venom (Marcussiet al., 2011), also neurotoxic, but at lower concentrations.

Conclusions:Genotoxic studies with animal venoms andtoxins are necessary because some venommolecules servedas templates for the preparation of pharmaceuticals andcountless others are being investigated for this purpose.

Keywords: Micrurus corallinus, comet, micronucleus.10.1016/j.toxicon.2012.04.010

10. Comet Assay and Micronucleus Tests to AssessDamage to the DNA of Human Lymphocytes Induced bythe Bothrops jararaca Venom

Silvana Marcussi 1, Marcus V.C. Trento 1,Mateus W. Eleutério 1, Marcel J. Palmieri 21Department of Chemistry, Federal University of Lavras - UFLA, MG, Brazil2Department of Biology, Federal University of Lavras - UFLA, MG, BrazilE-mail address: [email protected] (M.V.C. Trento).

Background:Bothrops jararaca is a species foundmainly inBrazil, but canalsobe found inotherSouthAmericancountriessuchasParaguayandnorthernArgentina. Thereareno reportsabout the impact of its venomon the DNA of animal cells and,thus, the objective of this study was to evaluate the genotoxic

potential of crude venom on human lymphocytes, in vitro,using the micronucleus test and comet assay technique.

Methods:Whole bloodwas incubated at different venomconcentrations for thepurpose of investigating the formationof micronuclei in binucleated cells using optical microscopy,after blocking cytokinesis with cytochalasin B (micronucleustest). The comet assay consisted of analysis of DNA frag-mentation by means of electrophoretic run for subsequentobservation of comets using a fluorescence microscope.

Results: The venom of B. jararaca induced the formationof micronuclei considering untreated blood, but thenumber of micronuclei formed was less than that observedin the positive control group (cisplatin), even at a venomconcentration 5 times higher than that of the control drug.The cytokinesis-block proliferation index (CBPI) wassimilar for treated and untreated cells, i.e., there was nochange in cell growth. Various levels of cell damage couldbe observed during the comet assay, especially level 2(20-40%) and level 3 (40-85%). Increased levels of celldamage could be observed in the same assay after doxo-rubicin has been used as positive control.

Discussion: The results of the comet assay demonstrateDNA strand breakage induced by the B. jararaca venom,mostly of local toxic action, as observed by Marcussi et al.(2011) for the Crotalus d. terrificus venom, whose action isprimarily neurotoxic. The results suggest that different toxinssuch as peptides, phospholipase A2, proteases, L-amino acidoxidase and hyaluronidase may have genotoxic potentialthrough distinct mechanisms, which are still unknown.

Conclusion:The fact of this venomhasbeen able to inducedamage to theDNAof human cells, however, to a lesser extentthan that observed for antitumor drugs, emphasizes the needfor further studies to characterize in a broadly manner theeffects of molecules isolated from venoms, aiming at theirfuture application for the development of new drugs, poten-tially more specific and less harmful to the human body.

Keywords: Bothrops jararaca, comet, micronucleus, human lymphocytes.10.1016/j.toxicon.2012.04.011

11. Effect of Zingiber officinale Plant Extract on theDifferential Control of Growth, Apoptotic Activity andGene Expression in Human Breast Cancer Cells

Ayman I. Elkady 1,2, Osama A. Abuzinadah 1,Nabih A. Baeshen 1, Tarek R. Rahmy 1,3

1Department of Biological sciences, Faculty of Science, King AbdulazizUniversity, Jeddah, Saudi Arabia2 Zoology Department, Faculty of Science, Alexandria University,Alexandria, Egypt3 Zoology Department, Faculty of Science, Suez Canal University, Ismailia, EgyptE-mail address: [email protected] (N.A. Baeshen).

Objective: The present study aimed to examine theability of themethanol extract of themedicinal plant ginger(Zingiber officinale) from Saudi Arabia, to inhibit theproliferation and colony formation in breast cancer celllines, MCF-7 and MDA-MB-231.

Methods: Cell viability, colony formation, dna frag-mentation, apoptotic assays, and reverse transcription-pcrand western blot analysis were used in the study.




Results: Ginger treatment resulted in dose-dependentsequences of events marked by apoptosis, as shown by lossof cell viability, chromatin condensation, DNA fragmenta-tion, activation of caspase 3, and cleavage of poly (ADP-ri-bose) polymerase. At the molecular level, the apoptotic celldeath mediated by ginger could be attributed in part to up-regulation of Bax and down-regulation of Bcl-2 proteins.Ginger treatment modulated expression of proteinsinvolved in cell cycle regulation; it decreased expression ofcyclin D1, cyclin-dependent kinase-4 (CDK-4), butincreased expression of CDK inhibitor, p21. It also inhibitedthe expression of the two prominent molecular targets ofcancer, c-Myc and the human telomerase reverse tran-scriptase (hTERT).

Conclusion: These findings suggest that the ginger maybe a promising candidate for the treatment of breastcarcinomas.

Keywords: Zingiber officinale, medicinal plants, apoptosis, gene expression,breast cancer, cell line10.1016/j.toxicon.2012.04.012

12. Structural Interpretation for the SubnanomolarAffinity of Muscarinic Toxin 7 for Human MuscarinicAcetylcholine Receptor 1 by Modeling Protein-ProteinInteraction

Jianrong Xu 1, Jun Xu 2, Hongzhuan Chen 1

1Department of Pharmacology, Institute of Medical Sciences, Shanghai JiaoTong University School of Medicine, Shanghai, P. R. China2Research Center for Drug Discovery, School of Pharmaceutical Sciences,Sun Yat-Sen University, Guangzhou, P. R. ChinaE-mail address: [email protected] (J. Xu).

Background: Human muscarinic acetylcholine receptor1 (hM1) is closely related to several diseases, such as Alz-heimer's disease, schizophrenia, and peptic ulcer. MT7,a muscarinic toxin isolated from the snake venom ofDendroaspis angusticeps, is the only natural selective hM1allosteric binder with subnanomolar affinity (Kd ¼ 14pM).MT7 is a peptidewith 66 residues, possessing a three-fingerstructure; and hM1 is a GPCR membrane protein, sharingthe conserved structure of seven transmembrane regions.Understanding the binding mode of hM1- MT7 will giveinsights to discover small molecular selective ligand forhM1.

Methods: The structure of hM1 was constructed byhomology modeling, and the bilayer membrane was createdby the VMD program. The initial interaction coordinate ofhM1-MT7 was generated by protein-protein docking inPatchDock program. Explicitmembranemolecular dynamics(MD) simulations with Amber program were utilized toproduce the dynamic trajectories of hM1-MT7. Bindingenergy between hM1 and MT7 was calculated with MM/PBSA method and decomposed into every residue to revealthe binding mode of hM1-MT7.

Results and Discussion: The hM1-MT7 binding mode isdiscovered to consist of five residue interaction clusters,which are separated in three interaction regions. Byanalyzing the cluster representative structures, the clusterresidues form an interaction network, which showsa multiple-point-to-site binding mode. It is revealed in

hydrogen binding statistical analysis that Glu170 (hM1)and Arg34 (MT7) are both locked in electrostatic cages withcounter charges, respectively. It makes hM1-MT7 hard todissociate, leading to the high binding affinity of MT7. Thisis confirmed by the dynamic distances calculation betweenthese residues, and it is consistent with biological mutantexperiments.

Conclusions: MT7 is discovered to bind to hM1 ina multiple-point-to-site mode. By forming a core in theinteraction network, Glu170 (hM1) and Arg34 (MT7) areresponsible for the subnanomolar affinity, which is consis-tent with the biological experimental data.

Keywords: muscarinic toxin, muscarinic acetylcholine receptor, moleculardynamics simulations, protein-protein docking10.1016/j.toxicon.2012.04.013

13. Crotamine Pharmacology Revisited: Novel InsightsBased on the Inhibition of Kv Channels

Steve Peigneur 1, Diego Orts 2, Alvaro Prieto da Silva 3,Nancy Oguiura 4, Malvina Boni-Mitake 5, Eduardo Brandtde Oliveira 6, André Junqueira Zaharenko 3, J.C. de Freitas 2,J. Tytgat 11 Laboratory of Toxicology, University of Leuven (K.U. Leuven), CampusGasthuisberg O&N2, Herestraat 49, P.O. Box 922, B-3000 Leuven, Belgium2Departamento de Fisiologia, Instituto de Biociências, Universidade de SãoPaulo, São Paulo, SP, Brazil3 Laboratório de Genética, Instituto Butantan, São Paulo, SP, Brazil4 Laboratório de Ecologia e Evolução, Instituto Butantan, São Paulo, SP, Brazil5Gerência de Radioproteção, IPEN, CNEN, São Paulo, SP, Brazil6 Faculdade de Medicina de Ribeirão Preto, Universidade de São Paulo,Ribeirão Preto, SP, BrazilE-mail address: [email protected] (S. Peigneur).

Background: Crotamine, a 5KDa peptide possessesa unique biological versatility. Not only its cell-penetratingactivity has become of clinical interest but moreover, itspotential selective anti-tumor activity is of great pharmaco-logical importance. Before, several studies have attempted toelucidate the exact molecular target responsible for thecrotamine-induced skeletal muscle spasm. The aim of thisstudy was to investigate whether crotamine affects voltage-gated potassium (KV) channels in an effort to explain its invivo effects.

Methodology/Principal findings: Crotamine wasstudied on ion channel function using the two-electrodevoltage clamp technique on 16 cloned ion channels (12 Kvchannels and 4 Nav channels), expressed in Xenopus laevisoocytes. Crotamine selectively inhibits Kv1.1, Kv1.2 and Kv1.3channels with an IC50 of w300 nM and the key amino acidsresponsible for this molecular interaction are highlighted.Our results demonstrate for the first time that the symptomswhich are observed in the typical crotamine syndrome mayresult from the inhibition of Kv channels.

Conclusions/significance: The ability of crotamine toinhibit thepotassiumcurrent throughKv channels unravels itas the first snake peptidewith the uniquemultifunctionalitysuch as cell penetrating, antitumoral activity and Kv channelinhibiting properties. The potent and selective Kv channelinhibitingproperties, as demonstrated in thiswork, canbeanadvantage for the use of crotamine or its derivatives as anti-tumor drug. This new property of crotamine might explain




some experimental observations and opens new perspec-tives of pharmacological uses.

Keywords: Crotamine, voltage-gated potassium channel, snake toxin,Crotalus durissus terrificus, anti-tumor drug10.1016/j.toxicon.2012.04.014

14. Evaluation of the Cytotoxic Activity of Rhinellaschneideri Toad Poison on Tumor Cells and on HealthyMononuclear Cells

Elisa C. Fornari Baldo 1, Cássio P. da Silva 2,Suely V. Sampaio 2, Eliane C. Arantes 11Departamento de Física e Química, Faculdade de Ciências Farmacêuticas deRibeirão Preto, Universidade de São Paulo, Ribeirão Preto, SP, Brazil2Departamento de Análises Clínicas, Toxicológicas e Bromatológicas,Faculdade de Ciências Farmacêuticas de Ribeirão Preto, Universidade de SãoPaulo, Ribeirão Preto, SP, BrazilE-mail address: [email protected] (E.C. Fornari Baldo).

Introduction: Amphibian poisons, especially fromAnura order, contain a variety of biologically activecompounds such as biogenic amines, cardiotonic steroids,alkaloids and peptides. Cardiotonic steroids have beenshown to induce apoptosis in a wide spectrum of tumorcell. However, the detailed molecular mechanisms ofinducing apoptosis are still unclear. The aim of this studywas the comparative evaluation of the cytotoxicity ofRhinella schneideri poison (Rsp) on tumor cell lines and onhealthy mononuclear cells.

Material andMethods: AMTT (3-[4, 5-dimethylthiazol-2-yl]-2,5-diphenyl-tetrazolium bromide) assay was used todetect cell viability using tumor cell lines B16F10 (MurineMelanoma Cells), HL-60 (Murine Acute PromyelocyticLeukemia Cells), HepG2 (Hepatocellular Carcinoma HumanCells), PC-12 (Murine Pheochromocytoma Cells) and PBMC(Human Peripheral BloodMononuclear Cells). The cells (5 x104 per well) were plated in 96-well plates and incubatedwith Rsp (5, 10, 20, 50 and 100 mg/mL) for 24 h. Then, theMTT was added (10 mL) and after 3 h of incubation at 37C�,in 5% of CO2, it was observed the production of formazancrystals by viable cells. The solubilization of crystals wasperformed by addition of DMSO (100 mL) and the absor-bance (DO) at 570 nmwas measured. The cell viability wascalculated by the equation: cell viability (%) ¼ (DO treatedgroup ⁄ DO control group) x 100%.

Results: In the presence of Rsp (5, 10, 20, 50 and 100 mg/mL) the cell viability (%) to HL-60 and B16F10 were 19.1,19.1, 19.0, 17.5, 20.4, and 40.3, 37.1, 38.2, 32.4, 34.8,respectively. The values obtained to HepG2 and PC-12 were82.4, 76.7, 79.2, 83.1, 84.2 and 72.8, 85.6, 93.4, 92.7, 76.9,respectively. The cell viability to PBMC was also high (87.4,77.2, 81.3, 95.6, 99.7).

Discussion: TheMTTassays showed that the tumor celllines HL-60 and B16F10 are more sensitive to Rsp, whichinduced a marked inhibition of cell proliferation. The celllines HepG2 and PC-12 showed high cell viability,meaning that Rsp slightly interfered with the replicationprocess of these cells. Rsp showed no cytotoxicity to PBMCcells.

Conclusion: These results show that Rsp has selectivecytotoxic activity for the different tumor cells evaluated,

indicating the potential application of its components astherapeutic agents in oncology.

Financial support: CAPES, CNPq, FAPESP.

Keywords: Rhinella schneideri, cardiotonic steroids, cell viability, anti-canceractivity10.1016/j.toxicon.2012.04.015

15. New Perspective for Therapy Against Seizures UsingMolecules From Rhinella schneideri Toad Poison

Mateus Amaral Baldo 1, José Luiz Liberato 2,Lívea Dornela Godoy 2, Wagner Ferreira dos Santos 2,Eliane Candiani Arantes 1

1Departamento de Física e Química, Faculdade de Ciências Farmacêuticas deRibeirão Preto, Universidade de São Paulo, Ribeirão Preto, SP, Brazil2Departamento de Biologia, Faculdade de Filosofia, Ciências e Letras deRibeirão Preto, Universidade de São Paulo, Ribeirão Preto, SP, BrazilE-mail address: [email protected] (M.A. Baldo).

Background: The number and diversity of compoundsproduced by amphibians in their glands is surprisingly high.Parotoid gland secretions from toads are useful source ofchemical compounds with potential medical-pharmaceu-tical applications, among them biogenic amines, cardiotonicsteroids, alkaloids and peptides. The aim of this study was toisolate and characterize components from Rhinella schneideri(Rs) poison and evaluate its potential use for inhibiting CNSseizures.

Material and Methods: The soluble poison wassubmitted to chromatography in HPLC system using C2C18column. Five main fractions were obtained and Rs5 showedneuroprotective action. Male Wistar rats (250 g) were can-nulated in the right lateral ventricle, following stereotacticcoordinates. Rats were divided into groups (n¼6) and thecontrol animals received injections of saline (0.9%; i.c.v.)followed by the convulsants PTZ (80mg/kg,i.p.) or NMDA (20mg/mL, i.c.v.). Different concentrations of Rs5 (0.05, 0.1, 0.25,0.5, 1.0, 1.5 and 2.0 mg/mL; i.c.v.) were analyzed. Additionally,the lower concentration (0.05 mg/mL) was used concomitantwith carbamazepine (CBZ) (20 mg/mL – protected 50% of ratagainst seizures) for evaluation of synergism between thetwo drugs.

Results: Pretreatment with Rs5 at concentrations of0.5, 1.0, 1.5 and 2.0 mg/mL protected 40, 60, 84 and 100%of rats against tonic-clonic seizures induced by PTZ,respectively. Additionally, 50, 62, 62, 83 e 100% of ratstreated with Rs5 at concentrations of 0.05, 0.1, 0.5, 1.0and 2.0 mg/mL, respectively, were protected of seizuresinduced by NMDA. To evaluate the Rs5 toxicity ratswere submitted to the rotarod assay after injection ofRs5 (2 mg/mL e 6 mg/mL mg/mL) and ataxia was notobserved.

Discussion: Rs5 concentration able to inhibit seizures islower than the concentration of CBZ which promotes thesame effect, showing that it is a molecule with high neu-roprotective activity. Additionally, was demonstratedsynergism between CBZ (20 mg/mL) and Rs5 (0.05 mg/mL)showing 100% protection against seizures. Furthermore, atthe rotaroad assay no animal fell from the apparatus indi-cating that Rs5 did not cause motor impairment.




Conclusion: Taken together, these results show that Rs5can be considered a molecule with high potential fordevelopment of a new drug for seizures therapy.

Financial support: CNPq, FAPESP.

Keywords: seizures therapy, neuroprotective effect, Rhinella schneideri10.1016/j.toxicon.2012.04.016

16. Marine Pharmacology and the Late 2011 MarinePharmaceuticals Pipeline

Alejandro M.S. MayerDepartment of Pharmacology, CCOM, Midwestern University, Downers Grove,Illinois, USAE-mail address: [email protected].

Review: As the renaissance in the pharmacology ofmarine natural products continues (Glaser and Mayer,Biochemical Pharmacology 78:440-448, 2009), the status ofthe clinical marine pharmaceuticals pipeline was assessedin early 2012. There were five FDA-approved marine-derived drugs in the US market, namely cytarabine (Cyto-sar-U�, Depocyt�), ziconotide (Prialt�), eribulin mesylate(Halaven�), brentuximab vedotin (Adcertis�), and omega-3-acid ethyl esters (Lovaza�), while vidarabine (Vira-A�)was no longer available, and trabectedin (Yondelis�) beingEU-registered. As of January 2012 there were 11 marine-derived compounds in the clinical marine pharmaceuticalpipeline, which was recently reviewed (Mayer et al. Trendsin Pharmacological Sciences 31:255-265, 2010). Included inthe eleven marine-derived compounds were three newadditions, namely monoclonal antibodies conjugated tosynthetic dolastatin derivatives that were in either Phase I,Phase II or Phase III clinical trials. Finally, the preclinicalmarine pharmaceutical pipeline remained an active globalenterprise with researchers from several countries report-ing novel mechanisms of action for multiple marinechemicals (Mayer et al. Comparative Biochemistry andPhysiology C 153: 191-222, 2011). Thus the clinical andpreclinical pharmaceutical pipelines continued to bevery active in early 2012. (http://marinepharmacology.midwestern.edu/).

Keywords: marine-derived pharmaceuticals; pharmacology; drugdevelopment10.1016/j.toxicon.2012.04.017

17. Novel Marine Compounds in Studies of NicotinicAcetylcholine Receptors

Igor E. Kasheverov 1, Denis S. Kudryavtsev 1,Elena V. Kyukova 1, Tatyana N. Makarieva 2,Natalia K. Utkina 2, Valentin A. Stonik 2,Victor I. Tsetlin 1

1 Shemyakin-Ovchinnikov Institute of Bioorganic Chemistry, RussianAcademy of Sciences, Moscow, Russian Federation2 Pacific Institute of Bioorganic Chemistry, Far Eastern Branch of RussianAcademy of Sciences, Vladivostok, Russian FederationE-mail address: [email protected] (I.E. Kasheverov).

Background: Nicotinic acetylcholine receptors(nAChRs) belong to superfamily of Cys-loop receptors,

containing also glycine, GABA-A, 5HT3 and some others,and are the best characterized. Definite subtypes of nAChRsare shown to be in association with nicotinic addiction andchronic pain, as well as with different diseases (muscledystrophies, schizophrenia, Alzheimer's and Parkinson'sdiseases). This is a strong argument for the search of novelpotent and highly selective cholinergic ligands to distinctreceptor subtypes and for designing new ones on the basisof known compounds. In addition to such well developedsources of bioactive compounds as snake and Conusmollusk venoms, at present other marine creatures are paidgreat attention.

Methods: We characterized a series of low molec-ular weight compounds extracted from some seaanemones and ascidia for their ability to bind to glycineand different nicotinic receptors as well as to acetyl-choline-binding proteins (AChBPs), naturally-occurringspatial homologs of the extracellular ligand-bindingdomains of all Cys-loop receptors. Among them werealkaloids, polysulphites, sphingolipid- and steroid-likesubstances.

Results and Discussion: Some of them were foundpotent competitors of radioiodinated a-bungarotoxin inbinding to classical agonist binding sites of nAChRs,which was in a good accordance with results of prelim-inary computational modeling and docking. In contrast,some other compounds potentiated the radioligandbinding to definite targets. Functional activity of allcompounds was studied electrophysiologically on glycineand a7 nicotinic receptors expressed in cells revealingseveral effective blockers. The data obtained mean thatsea anemones and ascidia are the additional rich sourcesof Cys-loop receptors’ ligands which could be perspectivebiomarkers or drugs. The possibility of preparation ofpotent markers for the a7 nAChR and different AChBPswas demonstrated with the use of peptides designed onthe basis of a-conotoxin PnIA from Conus pennaceus.Three novel analogs were found to be highly effectiveand selective ligands and were successfully prepared ina radioactive form with retained potencies and specific-ities for the a7 nAChR and/or AChBPs.

Supported by grants: RFBR #12-04-01746, MCB RAS(V.I.T.) and FP7 Neurocypres.

Keywords: marine compounds, nicotinic receptors, radioactive ligands10.1016/j.toxicon.2012.04.018

18. Comparative Toxicity of Binase towards Tumor andNormal Cells

Hector A. Cabrera-Fuentes 1,2, Pavel V. Zelenikhin 2,Aleksei I. Kolpakov 2, Klaus T. Preissner 1, Olga N. Ilinskaya 2

1 Institute for Biochemistry, Medical School, Justus-Liebig University, Giessen,Germany2Kazan Federal University, Department of Microbiology, Kazan, RussianFederationE-mail address: [email protected] (A.I. Kolpakov).

Review: Due to their biological activity ribonucleases areable to become the basis for the development of novel drugsin malignant neoplasms therapy. The work characterizes


http://marinepharmacology.midwestern.edu/

http://marinepharmacology.midwestern.edu/




cytotoxic activity of Bacillus intermedius ribonuclease towardssolid tumor cells: pulmonary adenocarcinoma (A549), humanfibrosarcoma (HT1080) and murine glioma C6 (ATCC). Theenzyme possesses high cytotoxic effect on A549 and C6 cellsand does not at the same time inhibit proliferation of HT1080cells. This fact could be explained by the different expressionlevels of ras-oncogenes by the tested cell lines. Ribonucleasedid not show cytotoxicity towards human umbilical veinendothelial cells (HUVEC) in concentration range of 0.1-300mg / ml. The data obtained indicate that detection of certainoncogenes in tumor cell as markers of their susceptibility tobinase cytotoxic action is very promising.

Keywords: binase, cytotoxicity, ras-oncogenes, A549, C6, HT1080, HUVECcell lines10.1016/j.toxicon.2012.04.019

19. Purification and Determination of AntibacterialConstituent, L-Amino Acid Oxidase from Calloselasmarhodostoma and Ophiophagus Hannah

Sugita Kunalan 1, Jaya Vejayan 1, Parasakthi Navaratnam1,Wayne Hodgson 2

1 Tan Sri Jeffrey Cheah School of Medicine and Health Sciences, MonashUniversity Sunway Campus, Selangor Darul Ehsan, Malaysia2 Faculty of Medicine, Nursing and Health Sciences, Monash UniversityClayton Campus, Victoria, AustraliaE-mail address: [email protected] (W. Hodgson).

Background: The emergence and re-emergence ofdiseases in addition to appearance of superbugs such asvancomycin- and methicillin-resistant Staphylococcus aureushas exposed the urgent and dire need for alternative antibi-otics. Recent research has shown evidence of snake venoms,namely Ophiophagus hannah (King cobra) and Calloselasmarhodostoma (Malayan pit viper) exhibiting strong potentialantibacterial activity against Staphylococcus aureus.

Methodology: Antibacterial activity was assessedagainst 22 bacterial strains including resistant Acinetobacterbaumanii, Enterococcus faecalis, Klebsiella pneumoniae,methicillin-resistant Staphylococcus aureus (MRSA) andStaphylococcus aureus strains using a hole-plate diffusionmethod, whereby the inhibition zone of the venoms wascompared to conventional antibiotics. Following that, usinga combination of successive purifications and the antibac-terial assay (bioassay guided purification), the characteriza-tion of the active constituent was achieved throughgel filtration, ion exchange and affinity-binding chroma-tography. The homogeneity and identity of the enzyme wasdetermined by gel electrophoresis and mass spectrometry.

Results: The active antibacterial constituent for bothvenoms was determined to be a homodimeric enzyme,L-Amino Acid Oxidase (LAAO). SDS-PAGE analysis revealeda single band of the purified enzymewith amolecular weightof approximately 65kDaand66kDa fromOphiophagushannah(OH) and Calloselasma rhodostoma (CR), respectively. BothOH-LAAO and CR-LAAO showed positive and effective inhi-bition of several Gram positive bacteria includingmethicillin-resistant Staphylococcus aureus with a minimum inhibitoryconcentration (MIC) of less than 10mg/mL, whereas bothenzymeswere less effective in inhibitingmost Gram negative

bacterium, which required MIC values above 20mg/mL. TheMIC result consistently suggests that the antibacterial activityof OH-LAAO is more effective than CR-LAAO. MIC values forCR-LAAOhave not beenpreviously reported. As a comparison,suitable antibiotics were also tested and observed.

Discussion: Past studies have stated that specific bindingis necessary and potentially vital as the bacterial growthinhibition is achieved by high concentrations of H2O2 whenin close contact to bacterial surfaces. The MIC resultspropose that CR- and OH-LAAOs probably attach morestrongly to Gram-positive bacterial surface than to Gramnegative bacterial surface and are thereforemore effective ininhibiting Gram positive bacterial growth inhibition.

Conclusion: OH and CR-LAAO have effective antibacte-rial activity, especially against Gram positive bacteria. Thisfinding encourages further work to elucidate their potentialin the development of a novel antibiotic.

Keywords: Ophiophagus hannah (OH), Calloselasma rhodostoma (CR),L-amino acid oxidase (LAAO), methicillin-resistant Staphylococcus aureus(MRSA), antibacterial10.1016/j.toxicon.2012.04.020

20. CFTR as a New Target for Crotoxin: PotentialApplication for Cystic Fibrosis

Grazyna Faure 1, Naziha Bakouh 2, Frederick Saul 3,Haijin Xu 1, Gabrielle Planelles 2, Mario Ollero 2,Aleksander Edelman 2

1 Institut Pasteur, Récepteurs-Canaux, Dept. of Neurosciences, Paris, France2 INSERM U845, Paris, France3 Institut Pasteur, Plate-Forme 6 - Cristallogenèse et Diffraction des Rayons X,Paris, FranceE-mail address: [email protected] (G. Faure).

Background: Crotoxin is the major heterodimerictoxin from the South American rattlesnake (Crotalus dur-issus terrificus) possessing PLA2 activity. Crotoxin princi-pally exhibits pre-synaptic neurotoxicity throughinteraction with protein receptors, and displays beneficialproperties such as bactericidal, antitumoral and antiviralactivities. It has been shown that crotoxin also potentiatesL-type calcium currents and may offer potential clinicalapplication in the control of cardiac function. The aim ofthis study was to investigate the interaction of crotoxinwith the Cystic Fibrosis Transmembrane conductanceRegulator (CFTR), a cAMP activated Cl- channel whosedysfunction is responsible for cystic fibrosis, and to char-acterize the potential physiological relevance of thisinteraction.

Methods: We used X-ray crystallography to solve thethree-dimensional structure of crotoxin. For functionalstudies, two series of experiments were performed:(i) surface plasmon resonance (SPR) for kinetic interactionstudies and (ii) voltage-current (I/V) experiments in Xen-opus oocytes expressing CFTR. The latter allowed us todetermine cAMP-activated Cl- - CFTR currents measured inthe presence and absence of crotoxin.

Results: The crystal structure of crotoxin solved at 1.35Å resolution revealed key residues involved in the stabilityand toxicity of this potent heterodimeric beta-neurotoxin(Faure et al., 2011, J.Mol. Biol. 88, 69-76). Using SPR we




demonstrated a direct specific binding of crotoxin and itsPLA2 subunit to the nucleotide-binding domain (NBD1) ofCFTR. Using I/V measurements we detected an increase incAMP-activated Cl- - CFTR current when crotoxin or its CBsubunit were injected into Xenopus oocytes. Since thedeletion of Phe508 in NBD1 is the most frequent mutationleading to cystic fibrosis, we also studied the binding ofcrotoxin to the F508delCFTR mutant. We found that cro-toxin and its CB subunit effectively increase the channelcurrent, showing a potentiating or correcting effect onF508delCFTR.

Conclusions: This new crystallographic data and theunexpected beneficial effect of crotoxin on mutated CFTRprovide an original perspective to investigate the phar-macotherapy of cystic fibrosis.

Keywords: crotoxin, crystal structure, cystic fibrosis10.1016/j.toxicon.2012.04.021

21. Anti-endotoxin Effects and Pharmacology of theImmune Selective Anti-Inflammatory Derivatives(ImSAIDs)

Craig W. WoodsBioVeteria Life Sciences, LLC, Prescott, AZ, USAE-mail address: [email protected].

Background: Endotoxin is a frequent cause of morbidityin human medicine, often resulting in systemic immuneactivation and sepsis. Recently, scientists have characterizedthe peptide, phe-glu-Gly, derived from salivary proteins. feGexhibits potent and rapid anti-inflammatory and anti-endo-toxin properties, making it a potential drug developmenttarget for toxins that initiate the inflammatory response.

Methods: Analysis and summary of peer-reviewedliterature related to the immuno-pharmacology of thepeptide phe-glu-Gly (feG).

Results: feG attenuates the cardiovascular and inflam-matory effects of endotoxin and anaphylaxis by reducinghypotension, leukocyte chemotaxis, and vascular leak. feGaffects activated inflammatory cells, especially neutrophils,by regulating integrins and inhibiting intracellularproduction of reactive oxygen species. feG is active at lowdoses (100 mg/kg) and has a long (9-12 hour) biologicalhalf-life. Furthermore, feG has exhibited no toxicity instudies to date.

Discussion: The mechanism by which feG exerts itseffects is distinctly different from steroids or non-steroidalanti-inflammatories (NSAIDs). This newmechanism appearsto hold promise for therapeutic intervention of endotoxemiaand perhaps other toxins which affect vascular integrity andimmune activation.

Conclusions: As a biological therapeutic, feG holdspromise in toxicity or envenomations where and over-exuberant inflammatory responsesmay result. Further workis required to understand its applications in venom relatedevents such as tissue destruction, immune cell activation,vascular leak, and anaphylaxis.

Keywords: endotoxin, ImSAIDs, antitoxin10.1016/j.toxicon.2012.04.022

22. Engineering of an HB-EGF Inhibitor fromthe Diphtheria Toxin Receptor–Binding Domain forthe Treatment of HB-EGF-Related Diseases

Benoit Villiers 1, Sylvain Pichard 1, Alain Sanson 1,Stephanie Delluc 2, Bernard Maillere 1,Pierre-Louis Tharaux 3, Daniel Gillet 11Biology and Technology Institute of Saclay, Atomic Energy and AlternativeEnergies Commission, France2 Indicia Biotechnology, Lyon, France3UMR 970, Paris Cardiovascular Research Centre, Institut National de laSante et de la Recherche Medicale, FranceE-mail address: [email protected] (D. Gillet).

Background: HB-EGF is a growth factor ligand of theEGF receptors (EGFR) ErbB1 and ErbB4. It is involved inrapidly progressive glomerulonephritis, ovary andpulmonary cancer, vasospasm connected with cerebralcontusion, and perhaps diabetic retinopathy and reste-nosis. Inhibition of the HB-EGF - EGFR activation pathwaymay have strong therapeutic potency. However, treatmentwith chemical or antibody EGFR inhibitors lack specificityand lead to serious adverse effects. The membraneprecursor form of HB-EGF, pro-HB-EGF, is the naturalreceptor for diphtheria toxin. We designed a powerfulinhibitor of HB-EGF, both in its soluble and membrane-bound precursor forms by engineering of the diphtheriatoxin receptor-binding domain (DTR).

Methods: We combined structure modeling, sitedirected mutagenesis and directed protein evolution toimprove considerably the therapeutic potential of DTR.Three codon-biased libraries each covering 4 or 5 residues,mostly hydrophobic and potentially responsible for theinsolubility of the protein produced in E. coliwere screenedfor the generation of soluble mutants. Site-directed muta-genesis of the binding site of DTR was performed toenhance affinity. CD4 T-cell epitopes were identified byexperimental and in silico approaches and mutated todecrease immunogenicity of DTR. Antigenicity of theevolved DTRs was evaluated by ELISA using sera fromhealthy diphtheria toxin-vaccinated donors. Biologicalactivity of evolved DTRs was measured by their capacity toinhibit diphtheria toxin toxicity and HB-EGF dependent cellproliferation.

Results: DTR1 carrying mutations Y380K/Q387E/L390T/A395T was the most soluble mutant. Surprisingly, it is 60fold more affine for HB-EGF than native diphtheria toxin(Kdw 49 pM). DTR3 carrying the mutations F389Y andG510A in the binding site was 5 fold more affine than DTR1(Kdw 9.5 pM). DTR9 was derived from DTR3 by the intro-duction of the mutations N399K, V452T, V483Q, H492E,S494K, T517E and E497D (or T436H) destroying 12 mostdominant CD4 T cell. DTR9 was 4 fold more affine thanDTR3 (Kdw 2.2 pM). ELISA analysis of the sera of 20 healthydonors showed that most circulating anti-diphtheria toxinantibodies target the catalytic domain of the toxin and thatthe antigenicity of DTR9 is strongly diminished ascompared to the whole toxin.

Conclusion: DTR9 is a potent inhibitor of the solubleand membrane precursor forms of HB-EGF. Tenth of mg ofprotein are purified from one L of bacterial culture withoutoptimization. Solubility is greatly enhanced, affinity is 1400




fold higher than that of the parental toxin and immuno-genicity and antigenicity are greatly decreased.

Keywords: Diphtheria toxin, HB-EGR, therapeutic protein10.1016/j.toxicon.2012.04.023

23. Transformation of the Naturally Occurring Frog SkinPeptide, Alyteserin-2a into a Potent Anti-cancer Agent

J. Michael Conlon 1, Milena Mechkarska 1, Kholoud Arafat 2,Samir Attoub 2

1Department of Biochemistry, United Arab Emirates University, Al-Ain, U.A.E2Department of Pharmacology, Faculty of Medicine, United Arab EmiratesUniversity, Al-Ain, U.A.EE-mail address: [email protected] (J.M. Conlon).

Background: Cell-penetrating peptides are present inskin secretions of certain frogs and constitute a compo-nent of the animal's system of host defense that protectsagainst predators and pathogenic microorganisms. Thesecompounds have potential for development into potentanti-cancer agents especially for use against tumors thatcell have developed resistance to commonly used drugs.Alyteserin-2a (ILGKLLSTAAGLLSNL.NH2) is a cationic,amphipathic, a-helical peptide, first isolated from skinsecretions of the midwife toad Alytes obstetricans. Thepeptide displays moderate cytotoxic potency againstA549 human non-small cell lung adenocarcinoma cells(LC50 ¼ 80 mM) and human erythrocytes (LC50 ¼ 140mM).

Methods: Structure-activity relationships were investi-gated by synthesizing analogs of alyteserin-2a in which thehydrophilic amino acids in the peptidewere replaced by oneormore L-lysineorD-lysine residue. Theanti-tumoractivitiesof the peptides were determined in A549 cells by measure-mentof ATPconcentrations using aCellTiter-Glo luminescentcell viability assay during a 24 h incubation. Hemolyticactivities of the peptides were determined by measurementof the release of hemoglobin from human erythrocytes.

Results: The substitutions Ser7/ L-Lys, Gly11/ L-Lys,Ser14 / L-Lys and Gly11 / D-Lys in alyteserin-2aproduced analogs with increased (2 - 4 fold) cytotoxicpotency against A549 cells but the hemolytic activity of thepeptides also increased by a corresponding amount. Thesubstitution Asn15 / L-Lys produced the most potentanalog against tumor cells increasing the activity againstA549 cells by approximately 6-fold (LC50 ¼ 13 mM). Potencywas also increased 5-fold against HepG2 human hep-atocarcinoma cells (LC50¼ 19 mM), 4-fold against MDA-MB-231 breast adenocarcinoma cells (LC50 ¼ 14 mM), and4-fold against HT-29 human colorectal adenocarcinomacells (LC50 ¼ 30 mM). The hemolytic activity of [N15K]aly-teserin-2a (LC50 ¼ 50 mM) was 2.8-fold greater than thenative peptide. [N15K]alyteserin-2a is very soluble inphysiological media and relatively easy to synthesize.

Conclusions: Increasing cationicity while maintainingamphipathicity increases theanti-tumoractivityof alyteserin-2a.Althoughtootoxic itself for systemicuse, [N15K]alyteserin-2a is identified as a lead compound for the development offurther analogs with improved cytotoxicity against tumorcells, but decreased activity against non-neoplastic cells.

Financial support: This work was supported by theTerry Fox Fund for Cancer Research.

Keywords: anti-cancer, frog, peptide, cytotoxin10.1016/j.toxicon.2012.04.024

24. Recombinant Hybrid Proteins from CobraVenom Factor and Human C3: Promising Agents forTherapeutic Intervention in Complement-MediatedDiseases

David C. Fritzinger 1, Brian E. Hew1, Carl-Wilhelm Vogel 1,21University of Hawaii Cancer Center, University of Hawaii at Manoa,Honolulu, HI, USA2Department of Pathology, John A. Burns School of Medicine, University ofHawaii at Manoa, Honolulu, HI, USAE-mail address: [email protected] (C.-W. Vogel).

Background: Cobra Venom Factor (CVF) is the anti-complementary protein in cobra venom. Whereas it doesnot exert direct toxicity, it exhaustively activates comple-ment which is believed to help expedite the toxic venomcomponents to reach their targets through an increasedblood flow and vascular permeability at the site of enve-nomation as a consequence of the release of the comple-ment-derived anaphylatoxins C3a and C5a. CVF activatescomplement by forming a C3- and C5-cleaving enzyme, theC3/5 convertase, with prey complement factor B, analo-gously to C3b, the activated form of complement compo-nent C3. CVF and C3 (from all vertebrate species, includinghumans) have been shown to share extensive structuralhomology as evidenced by DNA sequence, amino acidsequence, immunological cross-reactivity, and crystallo-graphic domain structure. In contrast to C3b, which formsa rather short-lived convertase consistentwith its biologicalfunction of localized complement activation on a target cellsurface, the convertase formed with CVF is very stable andresistant to the complement regulatory proteins of the host.

Methods:We have created hybrid proteins of human C3with CVF by exchanging functionally important regions, orselected amino acid residues, in human C3 with the cor-responding regions or amino acid residues from CVF.

Results: The resulting hybrid proteins are human C3derivatives with the CVF-specific function of forming a stableconvertase in human serum, causing complement inhibitionby consumption. As adverse complement activation is animportant component of the pathogenetic process of manydiseases, thesenovelhybridproteins, referredtoashumanizedCVF, represent a novel class of complement-inhibiting agents.Inseveralpreclinicalmodelsofdisease, complementdepletionwith humanized CVF is a potent therapeutic approach,includingmyocardial infarctionreperfusion injury, age-relatedmacular degeneration, myasthenia gravis, and others.

Conclusions: In addition to being a novel therapeuticagent, hybridproteins of CVFandhumanC3 (or C3 fromotherspecies, including cobra) also represent important experi-mental tools to identify functionally important regions inCVF.

Keywords: cobra venom factor, complement, venom-derived drugdevelopment10.1016/j.toxicon.2012.04.025




25. Viability of Fibrin Sealant from Snake Venom asScaffold to Rat Marrow-Derived Mesenchymal Stem Cells

Vinicius Peron de Oliveira Gasparotto 1, Fernanda da CruzLandim e Alvarenga 2, Alexandre Leite Rodrigues deOliveira 3, João Ferreira de Lima Neto 1,2,3,4,Midyan Daroz Guastali 2, Leandro Maia 2,Gustavo Ferreira Simões 3, Benedito Barraviera 1,4,Rui Seabra Ferreira Jr. 1,41Botucatu Medical School, São Paulo State University (UNESP – Univ EstadualPaulista), Department of Tropical Diseases and Image Diagnosis, Brazil2 Faculdade de Medicina Veterinária e Zootecnia – Unesp / Botucatu;Departamento de Reprodução Animal e Radiologia Veterinária, Brazil3Universidade Estadual de Campinas – UNICAMP / Campinas, Instituto deBiologia, Departamento de Anatomia, Brazil4Center for the Study of Venoms and Venomous Animals (CEVAP), UNESP – SãoPaulo State University, BrazilE-mail address: [email protected] (R.S. Ferreira).

Background: The aim of this study was to evaluate thein vitro viability of biomaterial Fibrin Sealant (FS) derivedfrom snake venom as a scaffold for Rat marrow-derivedmesenchymal stem cells (MSCs). The FS is characterized asa biological adhesive material, and is produced and wassupplied by the Center for the Study of Venoms andVenomous Animals, CEVAP, Brazil.

Methods: Stem cells were collected from the bonemarrow of femurs and tibias of mice 30 days old. MSCswere characterized using flow cytometry with CD 44 andCD 90 MSC positive markers and MSC and CD34 (mono-nuclear stem cells) negative marker. To confirm the char-acterization of the MSCs, cultivations of the first pass wereused to differentiate MSCs into specific cell lineage (oste-ogenic, chondrogenic and adipogenic). To evaluate the invitro growth and cell viability with the biomaterial, inver-ted light optical microscopy, fluorescence microscopy andscanning electron microscopy and transmission were used.We also observed the spontaneous differentiation ability ofSF in contact with MSCs to osteogenic, chondrogenic andadipogenic lineage.

Results: Different microscopy showed that the SF usedwas able to accomplish the capture and maintenance ofMSCs. On further increased magnification, it was possibleto observe the cell interaction with adjacent cells andcellular extensions into the interior and surface of thebiomaterial. After eight days of preparation the confluenceof the cell culture was around 90% and the MSCs adheredto the surfaces of the sealant. Future studies will be per-formed to implement the fibrin sealant as a scaffoldenriched in vivo experimental models to investigate thetime required for mechanical support for the MSC toremain at the target site and to achieve the expecteddifferentiation.

Conclusions: The collection, cultivation and character-ization of rat MSCs were possible. The SF was effective asa biological scaffold and interacted with the MSCs keepingthem viable alternatives providing clinical and surgicaltherapies more efficient for regenerative processes.

Financial support: CAPES, FAPESP 09/06280-0.

Keywords: fibrin sealant, scaffold, snake venom, mesenchymal stem cells10.1016/j.toxicon.2012.04.026

26. nAChR Antagonist as Chemotherapeutic Agents ofCertain Lung Cancer Types Expressing CholinergicSystem. A Case of Snake Three-Fingered Toxins andAlkylpyridinium Polymers from Marine Sponge

Ana Zovko 1, Metka Filipi�c 2, Katja Kolo�sa 2,Tamara Lah Turn�sek 2, Tom Turk 1

1Department of Biology, Biotechnical Faculty, University of Ljubljana,Ve�cna pot111,1000 Ljubljana, Slovenia2Department of Genetic Toxicology and Cancer Biology, National Institute ofBiology, Ve�cna pot 111, 1000 Ljubljana, SloveniaE-mail address: [email protected] (T. Turk).

Background: The most common form of lung cancer isnon-small cell lung cancer (NSCLC). NSCLC have been shownto express molecules that are part of the cholinergic system,including a7 nicotinic acetylcholine receptors (nAChR). Nico-tine and acetylcholine act as agonists of a7 nAChRs provokingcell proliferation. It has been reported that exposure to nico-tine protects cancer cells expressing a7 nAChRs against theapoptosis induced by anticancer drugs. Antagonists of a7nAChRs can attenuate the proliferative effects of agonists andcould thus be considered as potential anticancer agents. Oneoption is well studied snake three-fingered neurotoxins i.e. a-bungarotoxin or a-cobrotoxin. Recently one of the syntheticalkylpyridinium polymers (APs) closely related to natural APsfrom marine sponge, which are cytotoxic for certain types ofcancercells,wasshowntobeastrongantagonistofa-7nAChR.

Methods: We have studied the survival of A549 adeno-carcinoma cells upon treatment by a-bungarotoxin,a-cobrotoxin and APS8 one of the APs synthetic analogues. Inaddition the APS8 were also used to study signaling path-ways, apoptosis and proliferation of A549 NSCLC cells andnormal cells. Evidence of apoptosis activation by APS8 inNSCLC was quantitatively analyzed by annexin V-FITC/pro-pidium iodide uptake analysis with fluorescent cytometry.Cell morphology of APS8 induced apoptosis was investigatedwith a combination of the fluorescent DNA-binding dyesacridine orange and ethidium bromide. Expression ofproteins upon treatment of cells by APS8 with or withoutnicotine was studied using Bio-Ray protein chips. We inves-tigatedmechanismunderlying the apoptotic activity of APS8.

Results: We have shown that a-bungarotoxin and a-cobrotoxin are about 50 fold less cytotoxic for A549 cells ascompared to APS8 analogue. APS8 has significant selectivecytotoxicity towards NSCLC cells, while has no influence onnormal lung fibroblast cells MRC5. Upon treatment withAPS, a large proportion of cancer cells undergo apoptosis,while normal cells are not affected. APS8 also attenuatesthe antiapoptotic effects of nicotine. Treatment of NSCLCwith APS8 upregulates proapoptotic proteins from Bcl-2family, while anti-apoptotic proteins are down-regulated.APS8 markedly increased the expression levels of deathreceptors. Our results imply that both intrinsic andextrinsic pathway of apoptosis are activated by APS8.

Conclusion: Apoptosis of cancer cells induced withnAChR antagonist APS8 may be a new and promisingmethod in lung cancer treatment.

Keywords: nAChR antagonists, cancer cells, apoptosis10.1016/j.toxicon.2012.04.027




27. Development of a Virtual System to Support ClinicalToxinology Research

Ana Silvia S.S.B.S. Ferreira 2, Benedito Barraviera 1,2,Silvia R.C.S. Barraviera 2, Luciana P.F. Abbade 2,Rui Seabra Ferreira Jr.1,2, Carlos A. Caramori 21Center for Study of Venoms and Venomous Animals (CEVAP), São PauloState University (UNESP), Brazil2Botucatu Medical School, Paulo State University (UNESP), BrazilE-mail address: [email protected] (R.S. Ferreira).

Review: This research aimed to develop a Virtual SupportSystem for Clinical Research for managing a clinical trial intoxinology. It will be deployed in the Clinical Research Unit(UPECLIN), Botucatu Medical School – UNESP. This unitparticipates in theNational Clinical ResearchNetwork (RNPC),along with 31 other centers in Brazil. The assembly of theproposal was needed because the current significance ofclinical research, the need to harmonize actions to teaching allcenters involved, and finally embed socially the researchsubjects and health professionals away from centers ofexcellence. Thus, this initiative will support a multicenter,controlled, randomized phase II clinical trial, which willexamine 260 patients with venous ulcers treated weekly for90 days, to test the topical use of fibrin sealant derived fromsnake venom. This systemwas designed to house coursewareto support research, conceptual informationanddata fromtheproject, in addition to ethical concepts, bioethical and telecare,for the research subjects. It also has teaching modules for on-line training of health professionals. This pathology isa common occurrence and when complicated by infection orchronic, is a serious public health problem. Therefore, thedevelopment of the virtual system to support toxinologyclinical studies was necessary to assist in the translation fromexperimental research to clinical trials, collaborating withdiseases of the same importance as well as shares of RNPC.

Financial support: Fapesp Proc 09/06280-0 [RSFJr];CNPQ Proc 563582/2010-3 [BB].

Keywords: virtual system, clinical trials, fibrin sealant, toxinology10.1016/j.toxicon.2012.04.028

28. Sea Anemone Peptides Modulate TRPV1 Activity andProduce Analgesia Without Hyperthermic Effect

A.Andreev Yaroslav, Irina V. Mosharova, Sergey A. Kozlov,Yulia V. Korolkova, Eugene V. GrishinShemyakin-Ovchinnikov Institute of Bioorganic Chemistry, Russian Academyof Sciences, ul. Miklukho-Maklaya, 16/10, 117997 Moscow, RussiaE-mail address: [email protected] (A.Andreev Yaroslav).

Background: The transient receptor potential vanilloid 1receptor (TRPV1) is a nonselective cationic ion channel. It canbe activated by a variety of stimuli: noxious heat (>43�C),protons, osmotic pressure, various lipid messengers and exo-genic ligands such as capsaicin, the pungent ingredient of chilipeppers. TRPV1 takes part in development and progress ofchronic pain and inflammation. Therefore, there is a consid-erable interest in identification and development of novelagonists and antagonists of TRPV1 for the treatment of thesepathological states. At present there are a lot of small organic

molecules that interact with the capsaicin binding site and inallostericmannerblockTRPV1activation. Clinical trails of suchmolecules revealed that TRPV1 involved in body temperaturecontrol and most of TRPV1 antagonists cause hyperthermia.

Methods: Electrophysiology, microplate readermeasurement of intracellular Ca2þ, single cell Ca2þ imaging,mice models of pain, core body temperature measurement.

Results: Earlier, analgesic polypeptides named APHC1,APHC2 and APHC3 were isolated from extract of seaanemone Heteractis crispa. We characterized these poly-peptides as potent TRPV1 modulators and compared theirpharmacology in various in vitro and in vivo models. Allpolypeptides partially blocked capsaicin-induced responseof receptors on CHO or HEK293 cells expressing TRPV1, butnone of them influenced on thermal activation. It is verylikely that sea anemone peptides APHC1-3 displayeddifferential activity on different modes of TRPV1 activation.APHC1-3 showed significant antinociceptive and analgesicactivity in vivomodels in reasonable doses (0.01-0.1 mg/kg)and did not cause hyperthermia. Intravenous administra-tions of polypeptides APHC1-3 prolonged hot-plate latency,blocked capsaicin-, acetic acid- and formalin-inducedbehavior and inhibited CFA-induced hyperalgesia.

Discussion:Newpolypeptide antagonists of TRPV1 causesignificant analgesia and no hyperthermia effect in experi-mental animals. Therefore, they could be a base formoleculardesign of TRPV1 antagonists with desirable properties.

Conclusions: Polypeptides APHC1-3 belong to newclass of TRPV1 modulators that produce significant anal-gesic effect without hyperthermic action.

Keywords: TRPV1, Hetractis crispa, analgesic10.1016/j.toxicon.2012.04.029

29.Russelobin, aNon-toxic Thrombin-likeSerineProteasefrom the Venom of Russell's Viper (Daboia russellirusselli): Possible Applications in Cardiovascular DrugDevelopment

Ashis K. Mukherjee 1,2, Stephen P. Mackessy 1

1 School of Biological Sciences, University of Northern Colorado, Greeley, CO, USA2Department of Molecular Biology and Biotechnology, Tezpur University,Tezpur, IndiaE-mail address: [email protected] (A.K. Mukherjee).

Background: Russell's viper venom is rich in proteolyticenzymes which affect the haemostatic system of an enve-nomated victim. However, relatively few proteases havebeen characterized from this venom. Here we report thepurification and characterization of a thrombin-like serineprotease from the venom of Daboia russelli russelli(Pakistan).

Methods: A Russell's viper thrombin-like enzyme(RVTLE) was purified from crude venom by size exclusionchromatography followed by reversed-phase HPLC andcharacterized for biochemical and pharmacological prop-erties, fibrinogen clotting activity and in vivo toxicity.

Results: RVTLE, named Russelobin, is a glycosylated,monomeric protein having amolecular mass of 51.3 kDa. Thedeglycosylated enzyme (after removal of N-linked sugars)showed a protein band of 22.2 kDa in SDS-PAGE. De novo





sequencing of Russelobin via LC/MS/MS analysis of trypticdigested peptides identified 5 unique peptides. An NCBI-BLAST search of these peptides against a snake venomprotein database revealed significant similarities to snakevenom thrombin-like enzymes and serine proteases. Russe-lobin hydrolyzed chromogenic substrates in the followingorder: plasma kallikrein>thrombin > factor Xa>trypsin>plasmin. Russelobin showed BAEE-esterase activity butwas devoid of TAME-esterase activity. The enzyme wassignificantly inhibited by serine protease inhibitors (AEBSF,benzamidine HCl) and a2-macroglobulin. Russelobin showedoptimum activity at 45 �C, pH 9.0 andwas very stable againstfive cycles of freeze-thawing. Russelobin was able to clothuman fibrinogen by cleaving the Aa chain of fibrinogen. TheBb chainwas degraded to amuch slower rate and the g chainof fibrinogen remained intact. HPLC analysis of fibrinopep-tides release pattern showed that Russelobin preferentiallyreleased FPA, and the release of FPB was observed only afterprolonged incubation of enzyme with fibrinogen. However,human thrombin released both the FPA and FPB to an equalextent. Plasmin-mediated degradation of fibrin formed bythe action of Russelobin was found to be significantly higherthan degradation of the fibrin clot formed by humanthrombin. Russelobin did not show cytotoxicity againstCOLO-205 and MCF-7 cells. The i.p injection of Russelobin atdose of 1 mg/kg body weight of mice was found to be non-toxic.

Discussion: Russelobin is only superficially similar tohuman thrombin and it results in plasma defibrinogenation.FPB is minimally cleaved, producing an easily (physically)disrupted clot which is rapidly degraded in vitro by plasmin.

Conclusion: The application of Russelobin in cardio-vascular drug development is indicated due to its lowtoxicity and its potential utility as a defibrinogenatingagent. We thank Kentucky Reptile Zoo for providing RVV.

Keywords: thrombin-like snake venom protease; Russell's viper; fibrinogenclotting protease; serine protease10.1016/j.toxicon.2012.04.030

30. Antimicrobial Peptides from Arachnid Venoms andtheir Biological Activity in the Presence of CommercialAntibiotics

Francia García 1, Elba Villegas 2, Gerardo Pavel Espino-Solis 3,Alexis Rodríguez 1, Jorge Paniagua-Solis 4,Lourival D. Possani 1, Gabriel Sandoval 4, Gerardo Corzo 1

1Departamento de Medicina Molecular y Bioprocesos, Instituto deBiotecnología, Universidad Nacional Autónoma de México, A.P. 510-3,Cuernavaca Mor., 62250, México2Centro de Investigación en Biotecnología, Universidad Autónoma del Estadode Morelos, Av. Universidad 2001, Cuernavaca, Morelos, México3Baylor Institute for Immunology Research, Dallas TX, USA4 Laboratorios Silanes S.A. de C.V., Col. Del Valle, México, D.F, MexicoE-mail address: [email protected] (G. Corzo).

Background: Since the development of penicillin in the1940s, the synthesis and use of different antibiotics have hadan important impact in human health. The emergence ofresistant strains has made bacterial infections graduallydifficult to treat with available antibiotics. Particularly,bacteria have developed different mechanisms for acquiring

or modifying their genes, an accelerate mechanism ofadaptation for survival of both, pathogenic and non-patho-genic organisms. The discoveries of some antimicrobialpeptides (AMPs) in spider and scorpion venoms as well as inother living organisms have driven the idea of using them asnew antibiotic tools. In this work we characterize chemicallyand prove the antimicrobial effects of two antimicrobialpeptides isolated from the spider Lachesana sp. and thescorpion Centruroides suffusus suffusus (C.s. suffusus) venoms.Additionally we present a new strategy of synergisticapproaches to improve their antibiotic capacity.

Methods: Lachesana sp. and C.s. suffusus soluble venomswere fractionated by HPLC. AMPs from these two venomswere screened using the zone inhibition test on agarcultures of Staphylococcus aureus. The arachnid AMPs werefurther purified and its primary structure sequenced byEdman degradation and mass spectrometry. The AMPswere evaluated with and without the presence of thecommercial antibiotics chloramphenicol, ampicillin, novo-biocin, streptomycin and kanamycin using a dilutionsusceptibility test in growth medium.

Results: Two AMPs were obtained, one from the venomof Lachesana sp. and other from C.s. suffusus, and namedLa47 and Css54 respectively. La47 (26 mer) is identical tothe AMP latarcin 3a from Lachesana tarabaevi, and Css54(25 mer) has a 41 % identity to the AMP Pin2 from Pandinusimperator. La47 and Css54 showed the largest synergiceffect by inhibiting the growth of Staphylococcus aureus orEscherichia coli in the presence of the commercial antibi-otics streptomycin and kanamycin.

Discussion: It is well accepted that the AMPs mode ofantibiotic action is through pore formation of the bacterialmembranes, which leads to membrane depolarization, andthen to diffusion of extracellular material into the bacterialcytoplasm. In this respect, streptomycin and kanamycincould find the way to the interior of the bacterial cell andinhibit the bacterial machinery of protein transcription,which would guide to bacterial death.

Conclusions: These data show a motivating outlook forpotential clinical treatments of bacterial infections usingmixtures of both AMPs and commercial antibiotics.

AcknowledgementsThis work was supported by grants from CONACyT CB-

2010-153606 and Laboratorios Silanes S.A. de C.V.

Keywords: venom, scorpion, antimicrobial10.1016/j.toxicon.2012.04.031

31. Spider-venom Peptides that Target the HumanNaV1.7 channel: Potential Analgesics for the Treatmentof Chronic Pain

Julie Klint 1, Raveendra Anangi 1, Mehdi Mobli 1,Oliver Knapp 2, David J. Adams 2, Glenn F. King 1

1 Institute for Molecular Bioscience, The University of Queensland, Brisbane,QLD, Australia2Health InnovationsResearch Institute,RMITUniversity,Melbourne,VIC,AustraliaE-mail address: [email protected] (J. Klint).

Background: Chronic pain is a major worldwide healthissue, with patients often receiving inadequate treatment.




The voltage-gated sodium (NaV) channel NaV1.7 has recentlybeen identified as a promising target for treatment ofchronic pain. Gain-of-function mutations in the SCN9A geneencoding the pore-forming a-subunit of NaV1.7 causepainful neuropathies whereas, loss-of-function mutationsresult in a congenital indifference to all forms of pain1. Thus,blockers of Nav1.7 are likely to be useful analgesics for thetreatment of persistent pain. However, it will be critical toensure that such blockers do not inhibit other NaV subtypeswith essential physiological functions, such as NaV1.5 that isrestricted to the heart and is critical for the rising phase ofthe cardiac action potential. Many venomous animals haveevolved toxins that modulate the activity of NaV channels.Spider venoms in particular are rich in NaV channel modu-lators, with one third of all known ion channel toxins fromspider venoms acting NaV channels.2

Methods: Based on their primary structure and cysteinescaffold, 12 distinct families of spider toxins that modulatethe activity of NaV channels (NaSpTxs) were identified3.Several of these families show activity at NaV1.7; one familyin particular is interesting from a pharmacologicallyperspective as somemembers display subtype selectivity. Inorder to determine the molecular epitopes that govern theinteraction of members of this NaSpTx family with NaV1.7and other NaV subtypes, we have developed a recombinantexpression system for several family members in order toproduce isotopically labeled peptides for NMR structuralstudies and point mutants for functional analyses.

Results: We have discovered that one member of thisNaSpTx family is the most potent blocker of NaV1.7discovered to date, and that variations in activity within thisfamily are correlated with subtle changes in the structureand dynamics of the peptide pharmacophore.

Discussion: Examining the potency and selectivity ofnaturally occurring sequence variants within this peptidefamily, as well as point mutants thereof, will enable us toprecisely determine themolecular epitopes thatmediate theirinteractionwithNaV1.7 aswell as key off-target NaV subtypes.

Conclusion: Structure-function relationship studies ofthis NaSpTx family will allow us to rationally designblockers of NaV1.7 with improved potency, selectivity, andanalgesic potential.

References

Cox, J.J. et al.(2006) Nature 444, 894–898.www.arachnoserver.org.Klint, J.K. et al. (2012) Toxicon. Submitted.

Keywords: spider-venom peptides, NaV1.7, therapeutics, analgesics,structure-function relationships, pharmacophore, drug development10.1016/j.toxicon.2012.04.032

32. Development of High Throughput CalciumChannel Assays to Accelerate the Discovery ofNovel Toxins Targeting Human Cav2.2 Channels

Silmara R. Sousa, Lotten Ragnarsson, Irina Vetter,Volker Herzig, Glenn F. King, Richard J. LewisDivision of Chemistry and Structural Biology, Institute for MolecularBioscience, The University of Queensland, St Lucia, Queensland, AustraliaE-mail address: [email protected] (S.R. Sousa).

Background: Voltage-gated calcium channels (Cav) aremultidomain membrane proteins that play essential rolesin the control of neurotransmitter release and nociceptivetransmission. Different Cav auxiliary subunits have beenshown to influence the pharmacological and physiologicalproperties of Cav, although the precise role of each subunitis not completely understood. Cav2.2, a validated analgesictarget, is potently and selectively inhibited by smallpeptidic toxins (u-conotoxins) expressed in cone snailvenoms. Some of these peptides have become usefulpharmacological tools, while others have shown potentialas therapeutic leads due to their specificity for Cav2.2.Ziconotide, a synthetic version of u-conotoxin MVIIA,inhibits Cav2.2 with high potency, and it is currently in themarket for treatment of severe intractable pain. Unfortu-nately, the poor selectivity of ziconotide and other availabledrugs still represent a challenge for the effective treatmentof chronic pain, which affects billions of people worldwide.Thus, a better understanding of the mechanisms involvedin pain pathways and the identification of novel and moreselective drug candidates is urgently needed.

Aims: The aims of this studywere to provide insights intothe contribution of Cav specific subtypes, and auxiliarysubunits, to the pharmacology of Cav antagonists, and thusprovide a molecular basis for the involvement of Cav2.2 inpain pathways; and to develop high throughput assays todiscover novel selective inhibitors for Cav2.2 from cone snailand spider venoms.

Key Results and Discussion: We identified the Cavsubtypes and auxiliary subunits endogenously expressed inthe human neuroblastoma SH�SY5Y cell line, and used Cavspecific blockers to pharmacologically characterize Cavchannels in these cells. These results allowed us to develophigh throughput radioligand binding and fluorescentcalcium imaging assays to accelerate the discovery andcharacterization of inhibitors of human Cav2.2. u-Con-otoxins, such as CVID, MVIIA and GVIA displaced the highlyselective radiolabeled peptide 125I-GVIA from SH�SY5Y cellmembranes with high affinity (pIC50 w 11). Surprisingdiscrepancies in toxin inhibition were found between thecell membranes and the whole cells in the binding assays,as well as in the calcium imaging assays, suggesting aninfluence of auxiliary subunits on u-conotoxin affinity.

Keywords: Cav2.2, calcium channels, SH-SY5Y cells, spider and cone snailvenom toxins10.1016/j.toxicon.2012.04.033

33. Mass Spectrometry as a Tool to Search SpecificLigands for G-Protein-Coupled Receptors

Camila T. Cologna, Julien Echterbille, Edwin de Pauw,Loïc QuintonLaboratory of Mass Spectrometry, University of Liège, Liège, BelgiumE-mail address: [email protected] (C.T. Cologna).

Background: G-protein-coupled receptors (GPCRs)constitute the largest family of transmembrane proteins,their importance arises from their role as signal transmittersand regulators of cellular response. They control almost allphysiological processes in humans and consequently they

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emerge as the molecular target of around 40% of thecommercial available drugs. However, only about 60 of 356GPCRs subtypes are targeted by those marketed drugs. The220 remaining GPCRs are still unexploited, mostly due to thelack of specific ligands. Since the number of bioactivecompounds found in animal venoms can reach up to 40millions, the use of this natural library as a source for newligands and pharmacological tools discoveries comes to light.Thisworkaimedat thedevelopmentand the improvementofa new mass spectrometry based technique able to identifynew ligands of vasopressin receptors type 2 (V2R), amemberof GPCRs superfamily.

Methods: Vasopressin receptors type 2, overexpressedin a commercial cell line, were incubated with vasopressinplus a simple mix of known peptides (e.g. bradykinin,angiotensin and P14R peptide). After the incubation, twofractions were obtained: one containing the peptides thatbound to the receptors (‘bound fraction’) and the secondthe peptides that not bound (‘free fraction’). Both fractionswere analyzed with a MALDI-TOF/TOF mass spectrometerto identify the peptides that bound to the V2R and to verifythat only the vasopressin was detected in the bound frac-tion. The second step of this work was linked to the incu-bation of V2R with crude (or fractions of) animal venomsnot only to ensure the workability of the establishedprotocol when applied for a complex mixture of unknowncompounds, but also to discover new specific ligands.

Results: The MS analyses showed the presence of the 3cited peptides in the free fraction, while in the boundfraction we just observed a peak correspondent to vaso-pressin. These results demonstrate the feasibility of theprotocol in fishing for specific ligands for V2R.

Conclusion: The present work developed and improveda new methodology to screen for specific ligands for V2receptors in either a simple mixture or a complex mixture,in consequence, this may assist in the discovery of newligands. Moreover this methodology may be used to findnew ligands for different subtypes of receptors or evencharacterize orphan receptors, since the protocol can beadapted for other GPCRs subtypes.

Keywords: MS, vasopressin receptors, venom10.1016/j.toxicon.2012.04.034

34. Miniaturization of m-Conotoxins as PeptidomimeticStrategy to Develop Selective Sodium Channel Blockers

Marijke Stevens 1, Steve Peigneur 1, Natalia Dyubankova 2,Eveline Lescrinier 2, Piet Herdewijn 2, Jan Tytgat 11 Laboratory of Toxicology; University of Leuven (KU Leuven), Belgium2 Laboratory of Medicinal Chemistry, Rega Institute for Medical Research,University of Leuven (KU Leuven), BelgiumE-mail address: [email protected] (J. Tytgat).

Review: In recent decades cone snail toxins(“conotoxins”) have become of great interest in the searchfor novel subtype-selective modulators of voltage-gatedion channels, including voltage-gated sodium channels(Navs). Malfunctioning of Navs is the underlying cause ofnumerous diseases and the availability of subtype-selectivemodulators of these channels is therefore of utmostimportance.

Methods:We studied novel, synthetic bioactive peptidesthat specifically act on Navs. They were designed startingfrom two known, naturally occurring conotoxins BuIIIC(from Conus bullatus) and KIIIA (from Conus kinoshitai). Bothtoxins have been shown to potently block VGSC isoforms.Based on their structure and on recent insights in thedevelopment of “cono- and peptidomimetics”, a series ofminimized peptides were created, consisting out of 12 to 16amino acids, with two disulfide bridges. After proper folding,mass and structure verification, their blocking activities ondifferent Nav isoforms (Nav 1.2-1.8) were investigated by twoelectrode voltage clamp.

Results: The most promising compound shows thefollowing IC50 v alues: 77.8 nM � 5.9 for Nav1.2, 53.4 nM �2.0 for Nav1.4, 2373.1 nM � 94.1 for Nav1.5, 115.7 nM � 27.4for Nav1.6, respectively. Its structure was determined byNMR spectroscopy, showing a scaffold that contains noa-helix but in which some of the key residues seem to besuperimposable with key residues of KIIIA.

Discussion: Recently, block of Navs (in particular Nav1.6and Nav1.2) was suggested as a novel therapeutic strategy inmultiple sclerosis. Therefore, we tested our miniaturizedcompound in an animal model of multiple sclerosis, experi-mental autoimmune encephalomyelitis. Surprisingly, ourresults could not underscore this strategy sincemice towhichthe compound was administered, showed aworsening of thedisease. This was for example represented by higher meanclinical scores and a lower amount of phosphorylated axons,demonstrated by a series of immunohistological experiments.

Conclusions: In summary, our results show that it ispossible to design very small but potent and selectivepeptides based on known pharmacophores of m-con-otoxins. On the other hand, our in vivo studies witha specific blocker of Navs, suggest a complex and ambig-uous role of Navs in an animal model of multiple sclerosis.

Keywords: conotoxin, peptidomimetics, sodium channel10.1016/j.toxicon.2012.04.035

35. Development of Guanidinium-Toxin-Based SodiumChannel Blockers as PET Imaging Agents andTherapeutics for Diagnosing and Treating Pain

John Mulcahy 1, Justin Du Bois 1, David Yeomans 2,Sandip Biswal 3, Matthew Axtman 1, George Miljanich 4

1 Stanford University, Dept. of Chemistry, Stanford, CA, USA2 Stanford University, School of Medicine, Dept. of Anesthesiology, Stanford,CA, USA3 Stanford University School of Medicine, Dept. of Radiology, Stanford, CA, USA4 SiteOne Therapeutics, Redwood City, CA, USAE-mail address: [email protected] (G. Miljanich).

Review: Existing treatments for moderate-to-severepain, including opioids, are not always effective and sufferfrom a wide range of side effects, including potential foraddiction. Guanidinium toxins (GTxs) are potent, naturally-occurring small molecules that inhibit voltage-gatedsodium channels (Nav) by binding to the channel pore andblocking Na ion transit. Because existing Nav blockers(lidocaine, bupivacaine, etc.) already serve as clinicallyimportant anesthetics and analgesics, GTxs also have




potential as in that regard. In fact, GTxs have been testedsuccessfully in clinical trials for several indicationsincluding pain, chronic headache, and anal fissures. GTxsinhibit six of nine mammalian isoforms of Navs at lownanomolar concentrations. Significantly, the cardiac iso-form, Nav1.5, is resistant; greatly reducing the risk of car-diotoxicity compared with existing Nav inhibitors. We havedemonstrated that modification of GTxs through de novochemical synthesis allows the introduction of improvedpharmacologic properties. For example, novel GTxanalogues show local anesthetic and analgesic activity inrat nociception assays for as long as nine days. No toxicitywas observed at doses up to 15 times the minimallyeffective dose. We continue to evaluate the efficacy and PK-ADMET properties of our GTx lead compounds to guidedevelopment of a long-lasting GTx-based therapeutic thatoffers significant improvements over current treatmentsfor moderate-to-severe pain. GTx analogues also have thepotential to serve as diagnostic pain imaging agents.Accurate identification of chronic pain generators isa significant clinical challenge currently. Nav expression isenhanced in neuronal and inflamed tissues that exacerbatenociceptive activity. This feature can be exploited to imageand localize these pro-nociceptive tissues. Thus far, wehave successfully imaged significant increases in Navdensity in an injured nerve using a novel radiolabeled GTxanalogue, 18F-saxitoxin ([18F]STx), and PET-MRI ina neuropathic pain model (spared nerve injury; SNI). SNIrats received [18F]STx (i.v.) under anesthesia, and dynamicscans of right and left thighs were obtained for 30 minuteswith small animal PET. Significantly increased [18F]STxlabeling can be seen in the SNI nerve compared to controlside 10-20 minutes after injection. Blocking with ‘cold’[19F]STx eliminates such a difference between the rightand left nerves, confirming the specificity of the tracer. Theresults indicate that [18F]STx shows potential as a specificradiotracer for visualizing Nav channels in the setting ofneuropathic pain and for providing a clinical tool forobjective, image-guided therapies to treat chronic pain.

Keywords: analgesics, pain, imaging, guanidinium toxin, saxitoxin, sodiumchannels10.1016/j.toxicon.2012.04.036

36. Random Peptide Library Based on a SpiderNeurotoxin, and Utilization of the Library in in vitroEvolution Directed to GPCR Ligands

Tai Kubo 1,2, Seigo Ono 1, Tadashi Kimura 1,2,Suzuko Kobayashi 1, Tetsuro Kondo 1, Eriko Fukuda 1,Tatsuya Haga 3, Kimihiko Kameyama 1

1Natl Inst Adv Ind Sci Tech (AIST), Tsukuba, Japan2United Grad School Drug Discov and Med Info Sci, Gifu Univ, Gifu, Japan3 Inst Biomol Sci, Gakushuin Univ, Tokyo, JapanE-mail address: [email protected] (T. Kubo).

Background: In vitro evolution is one of the effectiveapproaches in protein engineering to obtain peptidesspecifically recognizing target molecule(s). We previouslysucceeded in generating IL-6 receptor (IL-6R) agonists andantagonists by in vitro evolution from a random peptide

library with a three-finger neurotoxin scaffold (Naimuddinet al, 2011). Peptides were initially selected by binding tosoluble portion of IL-6R, and then their physiologicalactivities were confirmed by cell-based assay. Solubleproteins could be mostly the target. In contrast, to applyin-vitro evolution techniques to membrane proteins, opti-mization for protein solubilization/reconstitution is criticaland is a major bottleneck in the process. To overcome theproblems, we have developed a new technique namedPERISS (intra periplasm secretion and selection) method,and successfully applied to select peptides targetingmuscarinic receptor m2 subtype.

Methods: (1) Construction of random peptide library;the peptide GTx1-15 (34 aa) was used as a template.Peptide domains located on the surface and the domainsproposed to be involved in target recognition were esti-mated. The corresponding regions in the GTx1-15 cDNAwere replaced by random nucleotides (NNS)n. (2) PERISSmethod; the peptides and the target m2 receptor wereexpressed in the periplasm and in the inner membrane ofE. coli, respectively. Interaction between the peptide andthe target, and the following selectionwere achieved in theperiplasmic space. The peptides interacting with the targetm2 were collected as binding complexes [peptide-target-E. coli]. The selected peptides were recombinantlyexpressed in E. coli, and purified. Binding activity of thepeptide to m2 receptor was measured by [3H]-NMSreplacement assay.

Results: The peptide library was constructed based ona neurotoxin GTx1-15, which we originally isolated from thespider venom and characterized as a T-type calcium channelmodulator (inhibitor cysteine knot; Ono et al, 2011). Struc-ture elements for ICK scaffoldweremaintained in the library,while potential diversity is 1015 in calculation.We performedthe PERISS method to screen from the library for peptidesselectively recognize m2 receptor. After six rounds of thePERISS, selected peptide sequences showed convergence.One of the peptides showed moderate affinity (Ki

app w300nM) and subtype selectivity to m2 receptor.

References

Naimuddin, M., et al.,Molecular Brain (2011), 4, Article 2: Ono,S., Kimura, T., Kubo, T., Toxicon (2011) 58, 265-76: Naimuddin,M., Kubo, T., Anal Biochem (2011) 419, 33-39: Kimura, T., Ono,S., Kubo, T., Intl J Peptides (2012) 2012, Article 731293.

Keywords: in vitro evolution, scaffold, peptide, neurotoxin, GPCR, threefinger, ICK10.1016/j.toxicon.2012.04.037

37. A Sea Anemone Toxin to Treat Autoimmune Diseases:ShK and its Analogs

Christine BeetonBaylor College of Medicine, Dept. of Molecular Physiology and Biophysics,Houston, Texas, USAE-mail address: [email protected].

Background: CCR7- effector memory T (TEM) lympho-cytes are involved in chronic inflammatory diseases such as




multiple sclerosis, type 1 diabetes mellitus and rheumatoidarthritis. These cells up-regulate the Kv1.3 potassiumchannels upon activation and these channels play a majorrole in TEM cell activation and function. Blockers of Kv1.3and other potassium channels are found in many venoms,including that of sea anemones. In 1995, Castañeda andcolleagues extracted a potent peptidic Kþ channel blockerfrom the Caribbean sea anemone Stichodactyla helianthusand named it ShK, for Stichodactyla helianthus Kþ channeltoxin.

Methods: We have used ShK as an in vitro and in vivoproof of concept for targeting Kv1.3 channels for thetreatment of autoimmune disease. We have also used thispeptide as a template for the development of highlyselective Kv1.3 channel blockers.

Results: Rationale modification of the peptide ShKhas yielded several potent and highly selective Kv1.3channel blockers, such as ShK-186. ShK-186 preferen-tially targets human and rat TEM cells in vitro and in vivo.It prevents and treats rat models of chronic inflamma-tory disease (delayed type hypersensitivity, experi-mental autoimmune encephalomyelitis – a model ofmultiple sclerosis, and pristane-induced arthritis – ananimal model of rheumatoid arthritis) without pre-venting clearance of acute infections or displaying overttoxicity.

Discussion: ShK-186 is currently undergoing pre-clinical trials for the therapy of autoimmune diseases.Dr. Shawn Iadonato will further discuss pharmacoki-netic and pharmacodynamic perspectives in anothersession.

Conclusions: ShK and its analogs represent a new classof immunomodulatory compounds for the treatment ofchronic inflammatory diseases.

Keywords: autoimmune disease, potassium channel, sea anemone toxin10.1016/j.toxicon.2012.04.038

38. Development of the Sea Anemone Toxin ShK-186 forthe Treatment of Autoimmune Diseases: PK and ADMEPerspectives

Christine Beeton 1, K. George Chandy 2, Shawn P. Iadonato 3,Ernesto Munoz-Elias 3, Eric J. Tarcha 3

1Department of Physiology and Biophysics and the Department of Surgery,UC Irvine, Irvine, CA, USA2Department of Molecular Physiology and Biophysics, Baylor College ofMedicine, Houston, TX, USA3Kineta Inc., Seattle, WA, USAE-mail address: [email protected] (S.P. Iadonato).

Background: ShK-186, a specific peptide inhibitor of theKv1.3 channel, is being developed for the treatment ofautoimmune diseases with a specific focus on multiplesclerosis. The Kv1.3 channel is critical to the proin-flammatory properties of effector memory T cells, andblockade of the channel diminishes the activation andexpansion of this cell population in vitro and in vivo. Thedrug is effective in reducing the clinical signs and histo-pathological correlates of disease in animal models ofMS, rheumatoid arthritis, psoriasis, skin hypersensitivity,

and autoimmune glomerulonephritis (see presentation byDr. Christine Beeton).

Methods: We have recently completed a series of IND-enabling nonclinical studies in rat and cynomolgusmonkeythat will be used to support first-in-human clinical trials ofShK-186. A key component of these studies was a thoroughcharacterization of the absorption, distribution, metabo-lism, excretion and pharmacokinetic properties of the drugin rodent and primate species. Bioanalytical methods weredeveloped (HPLC-MS/MS, ELISA) to measure ShK-186 levelsin plasma from rat and cynomolgus monkey. In addition, an111In-labeled DOTA conjugate of ShK was developed thatretained full channel blocking potency and that enableddetailed PET-scanning studies of peptide biodistribution invivo.

Results: ShK-186 shares several important propertieswith other charged venom peptides including biphasicslow absorption from the injection site and wide distri-bution to peripheral tissues. These properties result inpersistent whole blood concentrations of the peptideabove the Kd for Kv1.3 channel blockade for 5 days in ratand greater than one week in squirrel monkey. Explor-atory studies in the rat delayed-type hypersensitivityand chronic-relapsing experimental autoimmuneencephalomyelitis models demonstrate that doseadministration every 3 – 5 days provides maximumtherapeutic benefit. These data support approximatelyonce-weekly dose administration in human clinicaltrials.

Discussion: While >30 peptides are FDA-approvedproducts, the market potential of these molecules has beentempered by an expectation of frequent parenteraladministration. Charged venom peptides may alternativelyprovide durable pharmacological effects consistent withbetter patient tolerability and compliance and improvedcommercial success.

Conclusions: We have completed the IND-enablingnonclinical program for ShK-186. The nonclinical pharma-cological properties of the peptide support once-weeklyadministration in human clinical trials.

Keywords: sea anemone, autoimmune, ShK, multiple sclerosis, EAE,encephalomyelitis hypersensitivity, pharmacokinetic, ADME, metabolism,venom, peptide, drug10.1016/j.toxicon.2012.04.039

39. Discovery and Development of c-Conopeptides forthe Treatment of Pain

Richard J. LewisInstitute for Molecular Bioscience, The University of Queensland, Brisbane,AustraliaE-mail address: [email protected].

Background: A new class of small peptides (chi-con-opeptides MrIA and MrIB) were discovered in the venom ofthe moluscivorous cone snail Conus marmoreus that non-competitively inhibited the norepinephrine transporter(NET).

Method: Based on its novel mode of action and speci-ficity, MrIA was used as a lead molecule by Xenome Ltd.




Early studies in pain models revealed MrIA potentlyreversed neuropathic pain (allodynia) that developed inrats following loose ligation of the sciatic nerve.

Results: Extensive analoguing identified Xen2174 asa product candidate for the treatment of moderate tosevere pain based on its improved stability, wide thera-peutic index, and long‑lasting antinociception in rodentmodels of neuropathic and post‑surgical pain. Toxicologydata and a Phase I intravenous study supported the inves-tigation of bolus intrathecal (IT) doses of � 40 mg inhumans. A single bolus IT injection in a cohort of mixedoncology patients (N¼37) with chronic pain demonstratedthat Xen2174 was safe and tolerated at doses ranging from0.025 to 30 mg. Clinical data over 4 days post-treatmentsuggested a possible association between dose and reduc-tion in patient pain, especially in patients with a baselineVAS pain score � 40 out of 100.

Conclusions: The favourable clinical safety profile ofsingle IT doses of Xen2174, and an association with painreduction in this open-label study, support the continuedclinical investigation of Xen2174 for the relief of moderateto severe pain. Recent SAR and mutational studies confirmthat c-MrIA inhibits NET by binding to the mouth of thetransporter in the “occluded” state.

Keywords: conotoxin, pain, norepinephrine transporter10.1016/j.toxicon.2012.04.040

40. The Anti-cancer Activity of the Venom from SpiderMacrothele raveni in vitro and in vivo

Li Gao 1, Yongqing Shen 2, Jing Zhang 3, Chunyun Li 1,Liang Li 1, Baoen Shan 3, Baohua Zhao 1, Jinglin Wang 4

1College of Life Science, Hebei Normal University, Shijiazhuang, China2Hebei Medical University, Shijiazhuang, China3 The Fourth Hospital of Hebei Medical University, Shijiazhuang, China4 The State Key Laboratory of Pathogen and Biosecurity, Beijing Institute ofMicrobiology and Epidemiology, Beijing, ChinaE-mail address: [email protected] (J. Wang).

Background: The spider Macrothele raveni is distributedin the hilly areas of Guangxi Province, China. It has beenfound that the venom from the spider M. raveni containsa mixture of compounds with different types of biologicalactivity.

Methods: U251 glioblastoma cells were treated withthe spider venoms of different concentrations andtime. The anti-cancer activity of the venom fromM. raveni in vitro was analyzed by some functionalassays including lactate dehydrogenase (LDH) releaseassay, flow cytometry and western blotting. BALB/cnude mice were used in experiment of tumors inhibi-tion in vivo.

Results: The LDH release assay showed that the venomat concentrations of 10, 20, 40 mg/mL inhibited U251 cellproliferation and it affected cell viability in dose- and time-dependent manners using [3H]-methyl thymidine incor-poration assay. U251 cells treated with the venom wereaccumulated on G2/M and G0/G1 phase of cell cycle detec-ted by flow cytometry. Western blotting analysis indicatedone of the pharmacological mechanisms of spider venoms

was to activate the expression of p21 gene. In addition,In vivo examination of the inhibition of the size of tumors ofnude mice using the spider venom (1.6, 1.8, 2.0 mg/g mice)revealed that tumors size was significantly decreased fromcontrols by 21 days of treatment and at all points of analysisfor 7 week (P<0.01).

Discussion: Our data suggest that inhibition of thespider venom from M. raveni may be a potential drug forhuman glioblastoma carcinoma. To date there have been nostudies on examining the activity of anti-tumor of thevenom from the spider M. raveni.

Conclusion: The venom from the spider M. raveniinhibits the activation and differentiation of glioblastomacell and induces apoptosis.

Keywords: macrothele raveni, spider venom, U251 cells, anti-canceractivity10.1016/j.toxicon.2012.04.041

41. Anti-Inflammatory Effect of Honey Bee Venom onWistar Rats Induced Poly Cystic Ovarian Syndrome byEstradiol Valerate

Mohammad Nabiuni 1, Kazem Parivar 2, Bahman Zeynali 1,Azar Sheikholeslami 1,2, Latifeh Karimzadeh 1

1Department of Biology, Faculty of Basic Sciences, Tarbiat MoallemUniversity, Tehran, Iran2 School of Biological Sciences, University of Tehran, Tehran, IranE-mail address: [email protected] (L. Karimzadeh).

Background & Aims: Polycystic Ovarian Syndrome(PCOS) is a low grade inflammatory disease characterized byhyper androgenemia, hirsutism, endothelial dysfunction,pathological angiogenesis and chronic anovulation. C-reac-tive protein (CRP) is a protein found in the blood released byadipocytes, which plays a key role in adipocytes, which playsa key role in PCOS. Cyclooxygenase-2 is a key enzyme whichconverts arachidonic acid into prostaglandins and is trig-gered by inflammatory stimuli, such as cytokines. Itsexpression increases in PCOS. Honey bee venom (HBV)contains a variety of biologically active components havingvarious pharmaceutical properties. This study was designedto detect the possibility of HBV application as an anti-inflammatory therapeutic agent. In the present study, theanti inflammatory role of honey bee venom on expression ofCOX-2 and level of CRP, indexes of inflammatory, in Wistarrats with polycystic ovarian syndrome was investigate.

Methods: 1mg/100grB.W Estradiol Valerate (EV) wassubcutaneously injected to induce PCOS in mature femalerats (170�200 gram). After 60 days, HBV was administeredfor consecutive 10 days and its results in PCOS treatmentswere investigated. Rats were sacrificed and ovaries weresectioned to determine histomorphometrical and immu-nohistochemical evaluations in ovary and liver. Testos-terone and estradiol detected by Chemo LuminesanceImmuno Assay. In order to detect CRP, ELISA kit was usedand determines the absorbance by reading at 450 nm inthree groups of EV-induced PCOS, HBV-treatment andnormal intact animals.

Results: Thickness of theca layer, number and diameterof cysts and atretic follicles and levels of CRP significantly




decrease in HBV group comparing with PCOS group.Moreover, corpus luteum, as a sign of ovulation, wasobserved in HBV-treated ovaries which were obviouslyabsent in PCOS group. The immunohistochemical analysesfor cyclooxygenase-2 expressions showed a decreaseexpression of cyclooxygenase-2 enzyme in envelop layersof follicles in group 3.

Conclusions: Our results suggest that beneficial effectof HBV may be mediated by the inhibitory effect of HBV onCRP and COX-2 levels.

Keywords: polycystic ovarian syndrome, honey bee venom, Cyclo-oxygenase-2, C Reaction Protein, Wistar rat.10.1016/j.toxicon.2012.04.042

42. Effect of Conotoxins on GABAC Receptor

Elba Campos-Lira 1,2, Estuardo López-Vera 1

1 Instituto de Ciencias del Mar y Limnología, Mexico2 Posgrado en Ciencias Biológicas, Facultad de Ciencias. Universidad NacionalAutónoma de México, Circuito Exterior, Ciudad Universitaria, Coyoacán, D.F.,MéxicoE-mail address: [email protected] (E. López-Vera).

Background: Due to the lethal effects of toxinsproduced by Conus snails on human beings, the interest fortheir study has increased, not only for their great diversity,but for the quantity of compounds that form the neurotoxiccocktail as well. These toxins, commonly known as con-otoxins or conopeptides, modulate ionic channels andreceptors of the CEntral Nervous System. We know thatalpha- and psi-conotoxins modulate subtypes of nicotinicacetylcholine receptors (nAChR) and sigma-conotoxinsmodulate the activity of serotonin receptor (5-HT3). Both ofthese receptors belong to the Superfamily of cys-loopreceptors, which also includes glycine (GlyR) and gamma-amino butiric acid subtype A and C (GABAAR and GABACR)receptors.

Methods: This project consisted on an electrophysio-logical evaluation of the effects of venom fractions on theGABACR. The venom was isolated from Conus spurius. Thepurification of the crude venom was performed by HPLCusing a C18 analytical reverse-phase column Vydac C18(4.6 mm x 250 mm, particle size 5 mm, 300 Ångstrom poresize). The electrophysiological characterization was per-formed on the homomeric r1 GABAC subtype (GABAC r1)receptor expressed in Xenopus laevis oocytes. Each oocytewas clamped at -60mV with two-electrode system andwas constantly perfused with an electrophysiologicalsolution. GABA gated-currents were elicited with 1 mMgama-amino butyric acid (GABA) pulses. The neurotoxicfractions were applied to each oocyte in a 5-min staticbath.

Results: Only one fraction out of 25 had effect on GABA-elicited currents with a 58% of inhibition.

Discussion: This is the first evidence of activity on GABAreceptors by conotoxins.

Conclusions: This work helps to elucidate the func-tional mechanism of this receptor subtype.

Keywords: conotoxins two electrode technique, GABAC r1 receptors.10.1016/j.toxicon.2012.04.043

43. Melittin Peptide Kills Trypanosoma cruziEpimastigotes and Trypomastigotes Forms by DifferentCell Death Phenotypes

Camila M. Adade, Isabelle Ribeiro, Joana Pais,Thais Souto-PadrónInstituto de Microbiologia Paulo de Góes, Universidade Federal do Rio deJaneiro, Rio de Janeiro, RJ, BrazilE-mail address: [email protected] (C.M. Adade).

Background: The Apis mellifera venom contains severalcomponents with a wide variety of pharmaceutical proper-ties, such as melittin, which comprises 40-50% of the ven-om's dry weight. Previous studies demonstrated itsleishmanicidal, anti-microbial and anti-tumor activities.Chagas’ disease, caused by Trypanosoma cruzi, is estimatedto affect 16-18 million people in Central and South America,and the patients’ treatment is based on drugs such asBenznidazole, which exhibits toxic effects and rarely bene-ficial in the disease chronic phase. Therefore, the develop-ment of new chemotherapeutic agents from natural sources,is a lining research to be exploited. This study displays themelittin activity against T. cruzi (CLBrener clone) epi-mastigotes and trypomastigotes, and over the host cells.

Methods: Four-day-old culture epimastigotes werecultivated for 4 days in LIT medium containing 1.34, 2.68 and5.36mg/ml of melittin. Tissue culture trypomastigotes wereincubated inRPMImediumcontaining 0.1, 0.2 and0.4mg/mlmelittin for 1 day. Melittin effects on epimastigotes growthand trypomastigotes lysis was evaluated by daily countingwith a Neubauer chamber. The peptide effects over parasitesmorphology were analyzed by electron microscopy. Theparasites viability was evaluated by flow cytometry usingpropidium iodide (PI) staining. To test the melittin cytotox-icity to thehost cells, peritonealmacrophageswere treated ornot with 1 and 5mg/ml for 48h and examined byMTS assay.

Results: The treatment resulted in a reduction of para-sites number, in a dosis-dependent manner. The IC50 forepimastigotes inhibition growth is 2.44 � 0.39 mg/ ml andLD50 for trypomastigotes lysis is 0.14 � 0.05 mg/ ml. Epi-mastigotes presented between 70 to 99% PI-positivestaining, and trypomastigotes 62 to 69%. The ultrastructureled us to believe the occurrence of different programmedcell death pathways, where epimastigotes were killed byautophagy (e.g. structures suggestive of autophagosomes)and trypomastigotes by apoptosis (e.g. abnormal nuclearchromatin condensation and kDNA disorganization). Theformazan precipitate did not occurred in the macrophagestreated with 5 mg/ ml, with a significant absorbancereduction compared to untreated cells.

Discussion: Our data demonstrate that melittin waseffective against the epimastigote and trypomastigoteforms of T. cruzi, which displayed different death pheno-types, at concentrations non-toxic to host cells.

Conclusions: These findings confirmed the greatpotential of natural products, such as melittin peptide, asa source to screen e develop new drugs for the treatment ofneglected diseases, such as Chagas’ disease.

Keywords: Melittin, Apis mellifera venom, programed cell death, Chagasdisease, Trypanosoma cruzi.10.1016/j.toxicon.2012.04.044




44. Leishmanicidal Effects of a Phospholipase A2Isolated from Crotalus viridis viridis Snake Venom

Camila M. Adade 1, S. Fernandes Anne Cristine 1,O. Carvalho Ana Lúcia 2, Russolina B. Zingali 2,Thais Souto-Padrón 1

1 Instituto de Microbiologia Paulo de Góes, Universidade Federal do Rio deJaneiro, Rio de Janeiro, RJ, Brazil2 Instituto de Bioquímica Médica, Universidade Federal do Rio de Janeiro, Riode Janeiro, RJ, BrazilE-mail address: [email protected] (C.M. Adade).

Background: Treatment of Leishmaniasis, caused byLeishmania sp., a disease which afflicts millions of peopleworldwide, is based on drugs as amphotericin B, pentavalentantimonial and pentamidine, which exhibit toxic effects andlimited efficacy. Therefore the search for new drugs isa lining research to be exploited. Several toxins have beenused as therapeutic agents and pharmacological tools fordrug development. Snake venom phospholipases, exhibiteddifferent in vitro effects such as modulate cell proliferation,bactericidal activity and Plasmodium falciparum inhibitiongrowth. Crotalus viridis viridis (Cvv) phospholipase A2 (PLA2)is a 14 kDa, non-neurotoxic, basic single-chain myotoxin,previously described. This work is based in the Cvv venomPLA2, and its effects over L. amazonensis promastigotes.

Methods: The crude venom extract was loaded onto toa reverse phase analytical (C8) column using a highperformance liquid chromatographer. A linear gradient ofwater/acetonitrile with 0.1% trifluoroacetic acid was used.The peak contained the isolated PLA2 (confirmed by SDS-PAGE and mass spectrometry) was collected, lyophilized,resuspended in distilled water and its protein contentmeasured. L. amazonensis promastigotes were incubated inSchneider medium, with 0.3125 to 10 mcg/ ml PLA2, at24oC, and the effect on the cells proliferationwas evaluatedby counting with a Neubauer chamber, up to 72 h. Theparameter used to estimate proliferation inhibitionwas theIC50, which corresponds to the drug concentration thatinhibited 50% cell growth. Parasites viability was assessedby flow cytometry using propidium iodide (PI) labeling, andthe morphological alterations were examined by lightmicroscopy through Giemsa staining.

Results: The treatment caused a significant inhibition(32% to 82%) in the parasites growth, dosis- and time-dependent, soon thefirst24hours. Thedataobtainedallowedus toestimate the IC50/ 24hof2.50�1.42mcg/ml,decreasingto 0.77� 0.5 mcg/ ml after the final 72 h. The morphologicalalterations presented by treated parasites were rounded andunusual cell body shapes, with loss of membrane integrity,corroborated by the 96.6% of PI-positive labeling.

Discussion: We presented the employee of Cvv PLA2against L. amazonensis promastigotes, which inhibited theparasites in vitro growth. Further studies by electronmicroscopy are underway in order to detect the intracel-lular targets and the PLA2 effects over amastigotes forms.

Conclusions: This work presented that Cvv PLA2 can beobject of further research for a new leishmanicidal agent, aswe have looked forward to achieving.

Financial Support: CAPES, CNPq and FAPERJ.

Keywords: PLA2, Crotalus viridis viridis, Leishmania sp., Leishmaniasis10.1016/j.toxicon.2012.04.045

45. Carbon-13 and Nitrogen-15 Turnover in Serum ofBubaline Donors of Biological Material for Medical Use

Daniela A. Fossato da Silva 1, Natália P. Biscola 1,Renato M.F. Souza 1, Denis A. Caetano 1, Juliana C. Denadai 2,Maria M.P. Sartori 2, Evandro T. da Silva 2, Carlos Ducatti 2,Cyntia L. Martins 3, André M. Jorge 3, Lucilene D. dosSantos 1, Rui S. Ferreira Junior 1, Benedito Barraviera 1

1Centro de Estudos de Venenos e Animais Peçonhentos (CEVAP), Faculdadede Medicina, Univ. Estadual Paulista, Botucatu, SP, Brazil2Centro de Isótopos Estáveis (CIE), Univ. Estadual Paulista, Botucatu, SP, Brazil3 Faculdade de Medicina Veterinária e Zootecnia, Univ. Estadual Paulista,Botucatu, SP, BrazilE-mail address: [email protected] (D.A. Fossato da Silva).

Background: Toxicology is a fascinating area forpotential bioprospect molecules in the development of newdrugs. The synergism between the animal toxins and bio-logical materials can be used to formulate a newcompound. In this regard, the fibrin sealant produced byCEVAP (Centro de Estudos de Venenos e Animais Peçon-hentos) is part of this strategy. The knowledge of theassimilation of carbon and nitrogen isotopes in differenttissues or fractions shows the turnover, which suggests theincorporation of diet as a function of feeding time. Thetraceability involves biological samples that reflect the diet,allowing detection of the inclusion of animal meal in thefinal product. This methodmay prevent the transmission ofdiseases such as Creutzfeldt-Jakob disease, characteristic ofhuman prion, which is also transmitted by the action on themedical use of drugs or implants. The aim was to evaluatethe d13C and d15N turnover in serum of bubaline for thecertification of "green buffalo" as a donor of biologicalmaterial in the development of products for medical use.

Methods: Three young buffaloes (Murrah) were evalu-ated, during 117 days, under two different composition ofisotopic feeding: 1) Diet with vegetable protein source,constituted by C3 e C4 photosynthetic plants species (0-21days); 2) Diet with animal protein source, constituted bybovine meat and bone meal (22-117 days). Isotopic ratios ofcarbon (13C/12C) and nitrogen (15N/14N) were measured ina Mass Spectrometer Delta V Advantage Isotope Ratio MS.To quantitatively measure the speed of isotopes replace-ment, after determining the time interval, we used theexponential function of time (turnover), expressed byequation 1: d &13C/15N (t) ¼ d & 13C/15N (f) þ [d & 13C/15N(i) - d & 13C/15N (f)] e-kt.. The half life of Carbon-13 andNitrogen-15, provided 50% of each diet (t ¼ T), was calcu-lated by the equation 2: T ¼ ln 2/k.

Results: Isotopic values of carbon-13 were initiallyexpressed by d13C ¼ -12,93 � 0,20&, reaching values ofd13C ¼ -11,83 � 0,16& to 117 days. For nitrogen-15, thevalues ranged from d15N ¼ 7,43 � 0,45& to d15N ¼ 8,68 �0,24&. The half-life values were T¼ 11.7 days for carbon-13and T ¼ 14.6 days for nitrogen-15.

Conclusion: The evaluation time of 95 days is sufficientto incorporate the isotopic signal of diet supplementedwith animal protein, fundamental for the certification of"green buffalo" such a donor of biological material.

Keywords: animal toxins, stable isotopes, fibrin sealant10.1016/j.toxicon.2012.04.046




46. Biological Features of an EnantiomericAntimicrobial Peptide from Scorpion Venom

Daniel Juarez Lopez 1, Gerardo Corzo 2, Alexis Rodriguez 2,Elba Villegas 1

1Centro de Investigacion en Biotecnologia, Laboratorio de Estructura,Funcion, e Ingenieria de Proteinas, UAEM, Cuernavaca, Morelos, Mexico2 Instituto de Biotecnologia, Departamento de Medicina Molecular yBioprocesos, UNAM, Cuernavaca, Morelos, MexicoE-mail address: [email protected] (E. Villegas).

Background: Pin2 is an antimicrobial peptide (AMP)originally isolated and purified from the venom of the scor-pionPandinus imperator. It hasbeenproved tobehighlyactiveon disrupting bacterial and eukaryotic cell membranes. Pin2could be an original drug to be used as pioneering peptideantibiotic for topical infections;however, it is easily cleaved inthe presence of proteolytic enzymes. Therefore, to exploreinnovative derivatives of Pin2 an enantiomeric form waschemically synthesized. In this work we investigated somebiological characteristics of D-Pin2 in the presence of a clin-ical isolated pathogenic strain of Pseudomonas aeruginosa.

Methods: P. aeruginosa was clinically isolated from aninfected wound. It was microbiologically verified bymorphological, biochemical and molecular assays. P. aeru-ginosa samples were grown on King B liquid media for2 days to allow production of proteases. The supernatantswere separated by centrifugation at 4oC and filtered. Theprotein was precipitated using acetone. The proteolyticdegradation of D-Pin2 in the presence of trypsin, humanserum and protein extracts from P.aeruginosa was assayedfor 3,12, and 24h at 37oC. Native L-Pin2was used as control.The content of D- or L- Pin2 was followed by HPLC.

Results: D-Pin2 was found to be less hemolytic andmore efficient against Gram – and þ strains. However, theminimal inhibitory concentration in the presence ofP. aeruginosawas 50% higher than that of native L-Pin2, butD-Pin2 was resistant to proteolytic activity of trypsin andenzymes from human serum. It is known that P. aeruginosasecretes several proteases including protease IV and alka-line protease, among others. Therefore, when D-Pin2samples were incubated with precipitated extracts ofP. aeruginosa, it was found a decrease of 20, 50 and 100 % ofD-Pin2 at 3, 12 and 24h, respectively, indicating thatP. aeruginosa may contain enantio-selective proteases.

Discussion: It is assumed a possible degradation ofD-Pin2 by the activity of D-aminoacid dehydrogenase orracemase reported in P.aeruginosa. D-aminoacid dehydro-genase from P.aeruginosa is highly active on D-alanine andsome D-amino acids. P.aeruginosa extracts with D-amino-acid activity are not reported. Could there be the presenceof a protease degrading D-aminoacids?

Conclusion: D-Pin2 has less hemolytic activity and it ismore resistant to protease degradation; therefore, it couldbe a drug candidate for topical infections but will be lesseffective towards P. aeruginosa.

AcknowledgementsThis work was supported by grants from CONACyT CB-

2010-153606, CONACyT CB-2008-01-106949 and Red-Farmed.

Keywords: Pin-2, degradation, P. aeruginosa10.1016/j.toxicon.2012.04.047

47. Rational Development of Novel Leads from AnimalSecretion Based on Coagulation and Cell Targets

Ana Marisa Chudzinski-Tavassi,Kerly Fernanda Mesquita Pasqualoto,Linda Christian Carrijo-CarvalhoBiochemistry and Biophysics Laboratory, Butantan Institute, Av. Vital Brasil.São Paulo, SP, BrazilE-mail address: [email protected] (A.M. Chudzinski-Tavassi).

Review: Animal venoms and secretions are sources ofnovel pharmacologically active molecules of potential thera-peutic value. We have screened venoms aiming to discover,identify and isolate peptide molecules active in themammalian haemostatic system. This research initiativeyielded a portfolio of promising drug candidates comprisingLopap from bristles of the Lonomia obliqua caterpillar andAmblyomin-X from saliva of the Amblyomma cajennense tick.These novel recombinant proteins and synthetic peptidesturned out to be multifunctional molecules, which are pres-ently under different phases of development processes.Amblyomin-X is a potential candidate to treat cancer andmetastasis. Lopap is aprothrombinactivatorwhichbelongs tothe lipocalin family and displays serine protease-like activitywith procoagulant effect. It also induces cytokine secretionand antiapoptotic pathways in human cultured endothelialcells. A Lopap-derived peptide is capable of inducing collagensynthesis infibroblast culture and in theanimaldermis. In thiscontext is crucial to elucidate the multifunctional propertiesof those molecules and provide a further data integrationallowing the identification of potential targets as well aspossible mechanisms of action, and then the prediction ofnovel leads (new chemical entities, NCEs) as drug candidatesoptimizing their therapeutic effects considering the systembiology. In vitro and in vivo models reinforce therapeuticapplications. The in silico methods (docking, moleculardynamics simulations, protein homology modeling, quanti-tative structure-activity and/or structure-property relation-ships, chemometric analysis) will generate molecular andmathematical models to establish the mechanisms of actionhypothesisandsupport therationaldesignofnovel leadswithimproved pharmacokinetic and pharmacodynamic profiles.Lopap and Amblyomin-X have patents licensed to pharma-ceutical industries and are subjects of efforts to reach firmproofs of concept. Thenon-clinical phase studies are currentlyunderway for Amblyomin-X.

Financial support: FAPESP, INCTTOX-CNPq.

Keywords: lonomia, amblyomma, coagulation10.1016/j.toxicon.2012.04.048

48. Use of Connectivity Maps and Platform Technologiesfor Drug Lead/Biosimilar Discovery in Venoms

Jay W. Fox, Aramadhaka Lavakumar Reddy, Alyson Prorock,Bojan Dragulev, Yongde BaoUniversity of Virginia School of Medicine, Department of Microbiology,Immunology and Cancer Biology, Charlottesville, VA, USAE-mail address: [email protected] (J.W. Fox).

Background:Venoms have long beenmined as resourcesfor newdrugs or drug leads. Traditionally, drug discovery has





taken the approach of screening thevenom libraries based onspecific, pre-identified targets. Once a hit is detected, thevenom is subjected to fractionation to isolate thecompound(s) of interest. This approach has proven moder-ately successful, particularly with high throughput screenswith good targets; however, this approach essentially ispredetermined to find what you are looking for and missnovel activities not associatedwith the target. Novel genomicapproaches to drug discovery have recently been demon-strated to be useful for identifying drugs for repurposing forapplications directed at new diseases. One such approach isthe use of gene expression profiles of known bioactivecompounds and drugs informatically associated with thegene expression profiles of certain disease states. This hasbeen described as Connectivity Maps (1). In our study, wehave extended the principle of Connectivity Maps as a novelapproach for discovering bioactivities in venoms in a non-biased manner.

Methods: As a proof of concept we carried out geneexpression profiling of both Gila monster (Heloderma sus-pectum) venom, and a venom component Byetta (exenatide),a drug currently on the market to treat diabetes, followed byinformatic analysis using Connectivity Map databases. Simi-larly, we analyzed peptidomes from several snake venoms.

Results: Using Connectivity Mapping on Gila monstervenom we were able to identify activity in the venom con-nected with several diabetes drugs. Likewise, Byetta alsomapped to diabetes drugs thus suggesting that use of thisapproach would have identified the anti-diabetes activity inthe venom. Interestingly, the venom also mapped to severalhypotensive drugs, which is not surprising given the hypo-tensive action of envenomation on victims. Connectivitymapping of the peptidomes of several viperid venoms indi-cated interesting potential activities in those libraries aswell.

Discussion: The use of platform technologies such asgene expression profiling coupled with informatic analysesusing large databases has proven of recent value in repur-posing of drugs for different diseases. Similar approaches asdemonstrated in these studies suggest an alternative,complementary approach for screening venoms for novelactivities with the goal of identifying drug leads and/orbiosimilars for further development.

Conclusions: Use of Connectivity Mapping on Gilamonster venomvalidated this approach as a proof of conceptthat novel bioactivities in animal venomsmay be discoveredin a non-biased manner. (1) Lamb et al., Science, 2006.

Keywords: connectivity maps, platform technologies, drug discovery,biosimilars10.1016/j.toxicon.2012.04.049

C. Evolution of Toxins & Venom Glands49. The Origin and Evolution of Metalloproteinases inthe Venom of Snakes

Nicholas R. Casewell 1, Wolfgang Wüster 2,Simon C. Wagstaff 1, Camila Renjifo 1,Michael K. Richardson 3, Freek J. Vonk 3,Robert A. Harrison 1

1Alistair Reid Venom Research Unit, Liverpool School of Tropical Medicine,Liverpool, UK

2 Environment Centre Wales, School of Biological Sciences, Bangor University,Bangor, UK3 Sylvius Laboratory, Institute of Biology, Leiden University, Leiden, TheNetherlandsE-mail address: [email protected] (N.R. Casewell).

Background: Snake venom metalloproteinases(SVMPs) are a pathologically-important, oftenmajor, toxincomponent of snake venoms, particularly in the venoms ofviperid snakes. The SVMPs are members of the largemulti-locus adamalysin gene family alongside ADAM (adisintegrin and metalloproteinase) and ADAMTS (ADAMwith thrombospondin motifs) proteins. Here we discussthe evolution of SVMPs from: (i) their single ancestralrecruitment into the venom of advanced snakes, to (ii) thediverse structural and functional isoforms observed invenom today.

Methods: Phylogenetic analyses were used to recon-struct the evolutionary history of the adamlysins, witha focus on the SVMPs. Subsequently, the mode and tempoof SVMP evolution was analysed using ancestral sequencereconstructions, positive selection tests and macromolec-ular structure modeling.

Results and Discussion: The ancestral recruitment ofSVMPs into venom resulted from the duplication of anADAM28-like gene. Consequently, basal SVMPs are mostclosely related to reptilian ADAM28 and mammalianADAM28, ADAM7 and ADAM decysin-1 proteins. Notably,ancestral SVMPs exhibit complete conservation ofcysteine residues with their non-venom ADAM homologs,demonstrating that the gain and loss of cysteine residuesthought to be important for facilitating structuralchanges/post-translational modifications are the directresult of mutations following the recruitment of SVMPsinto venom. Following this recruitment event, novel SVMPdomain scaffolds have been generated in viperid snakes(P-II and P-I classes) through the duplication of SVMPgenes coupled with the action of positive selection. P-IIISVMPs first evolved into the P-II structure through thesingle evolutionary loss of the cysteine-rich domain,whilst multiple independent losses of the P-II disintegrindomain have resulted in convergent evolution of P-ISVMPs. In both instances of domain loss, adaptive evolu-tion is a major driving force – positive selection was foundto predominately act on amino acid residues predicted tobe surface-exposed on the molecular surface of new SVMPscaffolds.

Conclusions: These results highlight how changes tothe genetic structure of venom toxins can catalyze theaccelerated evolution of novel proteins and facilitate majorstructural and functional alterations. The generation ofdifferent molecular scaffolds (P-I, P-II and P-III SVMPs)encoded by the same multi-locus gene family appears tofacilitate protein neofunctionalization, whilst also pre-senting an evolutionary advantage through the retention ofmultiple genes capable of encoding functionally distinctproteins.

Keywords: snake venom metalloproteinases, molecular evolution, toxin,Serpentes, phylogenetics, positive selection10.1016/j.toxicon.2012.04.050



50. Tetrodotoxin in North-American Newts

Dietrich Mebs 1, Mari Yotsu-Yamashita 2

1 Institute of Legal Medicine, University of Frankfurt, Frankfurt, Germany2 Tohoku University, Graduate School of Agricultural Science, Division ofBioscience & Biotechnology, Aoba-ku, Sendai, JapanE-mail address: [email protected] (D. Mebs).

Background: The red spotted newts, Notophthalmusviridescens, from the eastern part of North-America andnewts of the genus Taricha, which are distributed along thewest-coast of the continent, are known to be poisonous dueto the presence of high concentrations of tetrodotoxin(TTX) in their skin, but also in all organs.

Methods: TTX and its analogues, 6-epiTTX and 11-oxoTTXwere analyzed inmethanolic extracts of newt specimens andof their organs by using post-column LC-fluorescent detec-tion. TTX was also localized in tissue sections using a mono-clonal antibody-based immunoenzymatic technique.

Results: TTX and its analogues were detected in varyingconcentrations in Notophthalmus viridescens newts inadults as well as in the terrestrial efts. When kept incaptivity, toxin levels decreased over the years, offspringstotally lacked TTX. In a newt population from Pennsylvaniamore than 50 percent were found to be infected withintestinal parasites (nematodes, trematodes, cestodes)which were positively stained for TTX by immunohisto-chemical technique. Moreover, insect predators such asmantids (Tenodera spp.) were observed occasionallyfeeding on the newts. In the Californian newt, Taricha tor-osa, only TTX was identified in the skin and internal organs.The toxin was also present in the eggs and larvae, whichwas histochemically confirmed. However, none of thetoxi�ns analogues could be detected.

Discussion: Variability of the toxin levels in the newts isa commonphenomenon ranging fromzero to extremelyhighconcentrations. The biogenetic origin of TTX is still a matterof discussion. An exogenous source of TTX via the food chainor its synthesis by symbiotic bacteria like in marine animalsmay explain its high variability in certain populations. Therole of TTX in protecting the newts from predation or para-sitism is questionable, because parasites and some predators(insects, snakes) may sustain or adapt to the toxicity.

Conclusion: The occurrence of a toxin like TTX inamphibians (newts, frogs, toads) offers many opportunitiesto study evolutionary, ecological and biosynthetic aspectsof a natural compound.

Keywords: tetrodotoxin, newts, toxin evolution10.1016/j.toxicon.2012.04.051

51. Evolution of a Neurotoxin from a Defensin

Shunyi Zhu 1, Steve Peigneur 2, Bin Gao 1, Jan Tytgat 21Group of Animal Innate Immunity, State Key Laboratory of IntegratedManagement of Pest Insects & Rodents, Institute of Zoology, ChineseAcademy of Sciences, 1 Beichen West Road, Chaoyang District, Beijing, China2 Laboratory of Toxicology, University of Leuven, O&N 2, Leuven, BelgiumE-mail address: [email protected] (S. Peigneur).

Review: Evolution of toxic proteins (toxins) representskey functional innovations convergently occurring in

phylogenetically diverse animal lineages. However, theevolutionary scenario of such innovations remains largelyunknown. Previous studies have shown high structuralconservation between scorpion neurotoxins affectingvoltage-gated potassium channels (VGPCs) (abbreviated asa-KTxs) and antibacterial insect defensins. Here we high-light an evolutionary role for a small deletion event takingplace in an insect defensin which results in a neurotoxic a-KTx. In comparison with insect defensins, a-KTxs lack anamino-terminal loop (n-loop) but possess two conservedresidues (LysCys4XaaAsn, underlined here) involved ina direct interaction with the pore region of VGPCs. Wefound that five insect defensins from Hemiptera andHymenoptera also contain the two functional residues atequivalent positions to a-KTxs and thus represent missingevolutionary links between these two classes of peptidefamilies. Deletion of the n-loop of one such peptide navi-defensin2-2 removes steric hindrance of peptide-channelinteractions and results in a VGPC-targeted neurotoxin(herein termed navitoxin) that selectively blocks threedifferent mammalian VGPC isoforms with nanomolar tomicromolar affinities. Mutations of the two crucial residuesdiminished or significantly decreased the affinity of navi-toxin on these channels, demonstrating that this defensin-derived neurotoxin binds to VGPCs in the same mannerwith a-KTxs. Taken together, these results indicate thatevolution of toxicity can be achieved by one small deletionevent. Our finding might also be important in consideringtoxicity of antibacterial defensins as drugs.

Keywords: defensin, voltage-gated potassium channel, neurotoxin10.1016/j.toxicon.2012.04.052

52. Divergence in the Biological Activity andComposition of Venom from Mackay (QLD) and Barossa/Adelaide (SA) Populations of Australian Pseudonajatextilis (Serpentes: Elapidae): An Important Role ofProcoagulants in Rodent Prey Incapacitation

Jure Skeji�c 1,2, Wayne C. Hodgson 2

1Department of Biochemistry and Molecular Biology, BIO21 Institute,University of Melbourne, Australia2Monash Venom Group, Department of Pharmacology, Monash University,AustraliaE-mail address: [email protected] (J. Skeji�c).

Background and Aims: The eastern Brown snakePseudonaja textilis has evolved extremely toxic venom tosubdue prey, which consists mainly of mice, rats, lizardsand frogs. A study on museum specimens found thatindividuals from Queensland grow larger in body size thanthose in South Australia, and that the proportion of endo-thermic prey consumed increases with snake body size(Shine, 1989). A more potent venom would confer advan-tage in preventing escape of a large rodent prey or an injuryto the snake. It was thus interesting to compare the bio-logical activities of the Queensland P. textilis venoms tothose of South Australia in rats to see if the venom fromQueensland would affect the physiological functions ofa rat more severely and more rapidly than the venom fromSouth Australia.





Methods: Sprague-Dawley rats were used as the preymodel. The phrenic nerve-diaphragm preparationwas usedto assess neurotoxicity, and a rat plasma clotting time assayto examine coagulopathic activity of the venoms (obtainedfrom Venom Supplies, SA). Proteomic profiling of venomcomposition was carried out by size-exclusion HPLC.

Results: Mackay P. textilis (QLD) venomwas found to bemore coagulopathic than venom from specimens from theBarossa and Adelaide region (SA), with extremely fast onsetof clotting. Surprisingly, venom from Mackay specimenswas found to be significantly less neurotoxic than the Bar-ossa/Adelaide venom.When compared to Australian taipanOxyuranus scutellatus and O. microlepidotus venoms, both P.textilis venoms were significantly more coagulopathic.Venom composition was found to differ greatly betweenthe Mackay and Barossa/Adelaide populations, withMackay venom having larger peaks in the high molecularweight region. In contrast, in the Barossa/Adelaide venom,lower molecular weight toxins were more abundant.

Discussion: Coagulopathic activity is strongly associatedwith rodent prey incapacitation. A higher coagulopathicpotency of the Queensland P. textilis venom has likelyevolved to incapacitate its large rat prey rapidly. Oxyuranusspecies have less coagulopathic venoms than P. textilis, butthey are known to deliver higher quantities of venom.Prothrombin activator of P. textilis is a highmolecular weightprotein and a large peak in this region of the Mackay profileis likely associated with its extremely strong procoagulantactivity. Larger peaks in the neurotoxin molecular weightregions of the Barossa/Adelaide samplemay contribute to itsmore potent neurotoxic activity.

Reference:Shine, R. (1989). Constraints, allometry and adaptation: FoodHabits and Reproductive Biology of Australian Brownsnakes(Pseudonaja: Elapidae). Herpetologica 45:195-207.

Keywords: Pseudonaja textilis, venom, biological activity, divergence, coa-gulopathy, prey incapacitation10.1016/j.toxicon.2012.04.053

53. Evolutionary Expansion of Venom Genes in the KingCobra Genome

Freek J. Vonk 1,2, Christiaan V. Henkel 3,R. Manjunatha Kini 4, Harald M.I. Kerkkamp1,Herman P. Spaink 1, Hans J. Jansen 3, S. Asad Hyder 1,Pim Arntzen 2, Guido E.E.J.M. van den Thillart 1,3,Marten Boetzer 5, Walter Pirovano 5, Ron P.H. Dirks 3,Michael K. Richardson 1

1 Leiden University, Institute of Biology, Sylvius Laboratory, Leiden, TheNetherlands2Netherlands Centre for Biodiversity Naturalis, Leiden, The Netherlands3 ZF-screens B.V., Niels Bohrweg, Leiden, The Netherlands4 Protein Science Laboratory, Department of Biological Sciences, NationalUniversity of Singapore, Science Drive 4, Singapore5BaseClear B.V., Leiden, The NetherlandsE-mail address: [email protected] (M.K. Richardson).

Background: Snake venom is a complex mixture ofproteins and peptides evolved to immobilize prey and deter

predators. The rapid evolution of venom toxins is part ofa continuous predator-prey ‘arms race’ that representsa classic model for studying molecular evolution. Snaketoxins are thought to evolve from normal physiologicalproteins through gene duplication and recruitment to thevenomgland. However, in the absence of genomic resources,these hypotheses have remained mainly speculative.

Methods: Using Illumina sequencing technology wehave produced a draft genome of an adult male Indonesianking cobra (Ophiophagus hannah). We have deep-sequenced a king cobra venom gland, accessory venomgland and pooled body organ transcriptomes to helpestablishing intron-exon boundaries and identify pseudo-genes. The genomic sequence data were first assembled denovo into contigs, which were subsequently oriented andmerged in scaffolds. Haploid genome size was estimatedusing flow cytometry to be around 1.36-1.59 Gbp. In total,we generated 41.2 Gbp (approximately 28x genomecoverage) of sequence data. Our assembled draft has anN50 contig size of 3,982 bp, and an N50 scaffold size of 226Kbp. The contigs sum to 1.45 Gbp, and the scaffolds (whichcontain gaps) to 1.66 Gbp.

Results: Comparative genomics revealed evidence oftandem duplication of toxin genes encoding physiologicalL-amino acid oxidase, cysteine-rich secretory proteins andmetalloproteinases, followed by recruitment through selec-tive expression in the venom gland. By contrast, nervegrowth factor toxins appear to have evolved by duplicationand dual recruitment, while hyaluronidase and phospholi-pase B evolvedby recruitment of existing physiological geneswithout further duplication, similar to acetylcholinesterase.We also identify 21 different three-finger toxin (3FTX) genesin the genome, suggesting amassive expansionof this family.We find a significant variation in the expression levels ofthese different 3FTX genes in the venom.

Conclusions: These data show that venomgenes originateand evolve through multiple distinct mechanisms. Thesesequences provide a valuable resource for studying rapidevolution of gene sequences and the evolution of recruitmentof genes to different tissues, and could help unravel themolecular basis of the evolution of new gene function.

Keywords: genome, king cobra, venom10.1016/j.toxicon.2012.04.054

54. Individual Venom Profiling of Crotalus durissusterrificus Specimens from a Geographically LimitedRegion: Crotamine Assessment and Captivity Evaluationon the Biological Activities

Airton Lourenço Jr.1,3, Camila Fernanda Zorzella Creste 3,Luciana Curtolo de Barros 1,3, Lucilene Delazari dosSantos 1,3, Daniel C. Pimenta 2,3, Benedito Barraviera 1,3,Rui Seabra Ferreira Jr.1,31Botucatu Medical School, São Paulo State University (UNESP – UnivEstadual Paulista), Department of Tropical Diseases and Image Diagnosis,Brazil2 Laboratory of Biochemistry and Biophysics, Butantan Institute, São Paulo,SP, Brazil3Center for the Study of Venoms and Venomous Animals (CEVAP), UNESP –

São Paulo State University, BrazilE-mail address: [email protected] (R.S. Ferreira).




Background: Crotalus durissus terrificus (Cdt) venommajor components comprise crotoxin, crotamine, gyroxinand convulxin. Crotamine exerts a myotoxic action, amongothers, but its expression varies even amid snakes from thesame region. Biochemical, enzymatic and pharmacologicalvariations of venoms may be associated with the geography,climate, gender, age, and diet, as well as captivity time andvenom extraction intervals. The present study aimed tocharacterize the Cdt venom from the Botucatu region, (SP,Brazil), by assessing its biochemical, pharmacological andenzymatic properties.

Methods: Venoms from newly captured snakes andalready-captured animals were characterized compara-tively to verify the sexual, environmental (length ofcaptivity) and ontogenetic variations that could influencethe venom composition. Protein concentration, SDS-PAGEand RP-HPLC were performed and the coagulant, toxic(LD50) and crotamine activities were assayed. IndividualSDS-PAGE analyses (315 samples) were performed and thebiological activities of the venom of 60 adults (captive andnewly captured males and females) and 18 newborns werecompared with the Brazilian Reference Venom.

Results: Crotamine was found in 39.7% (125/315) of thesamples, as determined by SDS-PAGE and RP-HPLC. Proteinconcentrationdiffered significantly betweenadults (75%) andnewborns (60%). RP-HPLC and SDS-PAGE analyses showedhighly variable protein concentration and copious crotoxinisoforms; however, the LD50 values decreased during thecaptivity time. Cdt venom biological activities were similaramong adult groups, but diminished during the captivityperiod.

Conclusions: The current findings demonstrate thatvenoms vary significantly in terms activity and proteinconcentration, despite originating from the same specie andregion.

Financial support: Support: CAPES, FAPESP 09/06280-0.

Keywords: snake venom; environmental variation; sexual variation;ontogenetic variation; Crotalus durissus venom.10.1016/j.toxicon.2012.04.055

55. Intraspecific Variation of Biological Activities inVenoms from Wild and Captive Bothrops jararaca

Eduardo Saad 1,3, Luciana Curtolo de Barros 1,3,Natalia Perussi Biscola 1,3, Daniel Carvalho Pimenta 3,4,Silvia Regina Sartori Barraviera Barraviera 2,3,Benedito Barraviera 1,3, Rui Seabra Ferreira Jr.1,31Botucatu Medical School, São Paulo State University (UNESP – UnivEstadual Paulista), Department of Tropical Diseases and Image Diagnosis,Brazil2Botucatu Medical School, São Paulo State University (UNESP – UnivEstadual Paulista), Department of Dermatology and Radiotherapy, Brazil3Center for the Study of Venoms and Venomous Animals (CEVAP), UNESP –

São Paulo State University, Brazil4 Laboratory of Biochemistry and Biophysics, Butantan Institute, São Paulo,SP, BrazilE-mail address: [email protected] (R.S. Ferreira).

Background: The venom of Bothrops jararaca iscomposed of complex mixture of molecules, mainly lectins,metalloproteinases, serinoproteinases, desintegrins,

phospholipases and peptides. This composition may varyaccording to the snake's age, sex and region of origin.

Methods: The present work evaluated individual vari-ation of Bothrops jararaca venom in the Botucatu region,Sao Paulo state, Brazil, through enzymatic, biochemical andpharmacological characterization, utilizing in vitro testsand biological assays. The activities were compared withthose of Brazilian Reference Venom (BRV). Proteinconcentration varied between adult and juvenile groups.

Results: The electrophoretic profiles were similar, withmolecular masses ranging between 25 and 50 kDa, but withintraspecific variations. RP-HPLC revealed protein concen-tration variations. Coagulant activity did not differ amongadult groups, but there was a large variation between juve-nile and BRV, which coagulated more intensely. Venomsfrom adults presented greater hemorrhagic activity; espe-cially males recently arrived from the wild. On the otherhand, the juvenile kept in captivity and adult males pre-sented more elevated values. Edematogenic activity verifiedan increase in edema in all groups. At the mean lethal dose(LD50), toxicity varied significantly between groups, beingthe venom from captive females three timesmore toxic thanthat from juvenile.

Discussion/Conclusions: The results of this study illus-trate the intra- and interspecific complexity present in snakevenoms, possibly represented in this case by ontogenetic,sexual and environmental variability in Bothrops jararacavenom. The data presented herein suggest a possible venomspecialization in light of the factors studied. Furthermore, itis proposed that Brazilian public health authorities trackdown deeply the constitution of the pooled venomemployed in the immunization of serum-producing animalssince. Given the large territorial extension of Brazil, thisvariability would require regional monitoring and evalua-tion of the efficacy of bothropic antivenom on treatment ofsnakebite and the permanent sequela observed.

Financial support: CAPES, FAPESP 09/06280-0.

Keywords: snake venom; environment variation; sexual variation; ontoge-netic variation; Bothrops venom.10.1016/j.toxicon.2012.04.056

56. The Evolutionary Origins of Monotreme Crural Glands

Emily Wong 1, Camilla Whittington 1,2, Tony Papenfuss 3,Stewart Nicol 4, Wesley C. Warren 5, Katherine Belov 1

1 Faculty of Veterinary Science, The University of Sydney, NSW, Australia2 Institute of Evolutionary Biology and Environmental Science, University ofZurich, Switzerland3Bioinformatics Division, The Walter and Eliza Hall Institute of MedicalResearch, Victoria, Australia4 School of Zoology, University of Tasmania, Australia5 The Genome Institute, Washington University School of Medicine, St Louis,MO, USAE-mail address: [email protected] (K. Belov).

Background: Monotremes (echidna and platypus) areegg-laying mammals. One of their most unique character-istics is that males have crural glands that are seasonallyactive. Male platypuses produce venom in significantquantities during the breeding season, presumably to aid incompetition against conspecifics. Platypus envenomation




of humans causes excruciating pain and prolonged swellingthat is unresponsive to conventional painkillers. The factthat male echidnas also produce secretions during thebreeding season was only discovered recently. Given thatechidnas are not able to erect their spurs, the compositionand purpose of these secretions remains to be determined.

Methods: We used Illumina sequencing to identifyhighly expressed genes in the crural gland of both species.

Results: Peptides identified which are likely to beresponsible for the unique symptoms of platypus enveno-mation included serine proteases, metalloproteinases, anti-microbials, cytokines and protease inhibitors. The echidnacrural gland transcriptome appeared markedly different tothe platypus with no correlation between the top 50 mosthighly expressed genes between the datasets. Gene ontologyterms associated with the top 100 most highly expressedgenes in echidna, but not platypus, showed functional termsassociated with steroidal and fatty acid production.

Conclusions: The non-aggressive behavior of theanimal during the breeding season, the structure of thespur, along with the histology of the echidna venom glandsuggests that echidna venom has a vastly different purposeto platypus venom. Our early results support that echidnacrural gland proteins may function in chemical communi-cation consistent with a role in reproduction and that overevolutionary time the echidna crural gland must have lostits venomous capabilities.

Keywords: platypus, echidna, evolution10.1016/j.toxicon.2012.04.057

57. Structural Characteristics and Evolution of A NovelVenom Phospholipase A2 Gene from Protobothropsflavoviridis

Takahito Chijiwa 1, Naoki Ikeda 1 , Haruna Masuda 1,Hiroaki Hara 1, Naoko Oda-Ueda 2, Shosaku Hattori 3,Motonori Ohno 1

1Department of Applied Life Science, Faculty of Bioscience andBiotechnology, Sojo University, Ikeda, Kumamoto, Japan2Department of Biochemistry, Faculty of Pharmaceutical Sciences, SojoUniversity, Ikeda, Kumamoto, Japan3 Institute of Medical Science, University of Tokyo, Oshima-gun, Kagoshima,JapanE-mail address: [email protected] (N. Ikeda).

Review: Protobothrops flavoviridis (Habu, Viperidae, Cro-talinae) inhabit the southwestern island of Japan. Avariety ofphopholipase A2 (PLA2) isozymes constitute major toxiccomponents of P. flavoviridis venom. A novel PLA2 gene,named PfPLA 6, was found in 6,328-bp NIS-1(5’)-a segmentpreceding 25-kbp NIS-1(3’) segment, which harbors fivePLA2 isozyme genes, in P. flavoviridis genome, and sequenced.Its open reading frame (ORF) of PfPLA 6 encodes basic PLA2with SLVQ sequence in N-terminus, which is specific for[Lys49]PLA2 subgroup, and Asp at position 49, which isspecific for [Asp49]PLA2 subgroups. Thus, PfPLA 6 seeminglyencodes a hybrid of [Lys49]PLA2 and [Asp49]PLA2. However,PfPLA 6 is a pseudogene because the protein encoded by itsORF and its corresponding cDNA have not been isolated.Comparison of the nucleotide sequences of 71 Viperidae(Viperinae and Crotalinae) venom PLA2 isozyme genes

including PfPLA 6, particularly focusing on characteristicdeletion of 12-bp segment called S1EX 1 or 55-bp segmentcalled S2EX 1 in exon 1 and interposition of 219-bp segmentcalled SINT2, being in accordwith short interspersednuclearelement (SINE), in intron 2, enabled us to propose a novelclassification of Viperidae PLA2 genes into three types, A(with S2EX 1 butwithout SINT 2), B (without S1EX 1butwithSINT 2), and C (without S2EX 1 but with SINT 2). It is thoughtthat A-type PLA2 genes including PfPLA 6 without SINT 2,which are all pseudogenes, are evolutionarily ancestral toB-type and C-type PLA2 geneswith SINT 2. This classificationalso clarified that Viperidae PLA2 genes are B type and Cro-talinae PLA2 genes are C type. Since eleven P. flavoviridis PLA2isozyme genes including four inactive genes hitherto char-acterized have both S2EX 1 and SINT 2, they are all catego-rized as C type. As PfPLA 6 is a pseudogene, an activeprototype of PfPLA 6 could be assumed to be the ancestralPLA2 gene. Phylogenetic analysis of P. flavoviridis PLA2isozymes also suggested that PfPLA 6 is ancestral to otherPLA2 genes. Putative evolutionary processes from this A-typeprototype PLA2 gene to descendent PLA2 genes were dis-cussed. This work will be published in “Bioscience, Biotech-nology, and Biochemistry” this year.

Keywords: Protobothrops flavoviridis, phospholipase A2 gene, classification,SINE, ancestral gene.10.1016/j.toxicon.2012.04.058

58. Functional Redundancy in Venoms is anEvolutionary By-Product

David Morgenstern 1, Ricardo C. Rodriguez de la Vega 2,Michael Ott 3, Glenn F. King 1, Bryan G. Fry 4

1 Institute for Molecular Bioscience, The University of Queensland, StLucia,QLD, , Australia2UMR 7138, Département Systématique et Evolution, Muséum Nationald'Histoire Naturelle, Paris, France3CSIRO Ecosystem Sciences, Acton ACT, Australia4 School of Biological Sciences, The University of Queensland, St. Lucia, QLD,AustraliaE-mail address: [email protected] (D. Morgenstern).

Background: Functional redundancy in proteins is a rarephenomenon, with gene duplicates typically evolving ondivergent molecular evolutionary trajectories. Venomsrepresent a rare case of functional redundancy, making it ofa great interest to the evolutionary biologist. A long-standing theory has been that toxin diversity is a result ofdirectional (positive) selection, with the reciprocal muta-tion-reaction process between prey and hunter being themajor evolutionary selection pressure. This ‘immediacy’hypothesis does not take into account historical processesthat affect venom evolution. We used the scorpion cysteinestabilized ab (CSab) toxins as a model for an investigation ofthe evolutionary forces that shape venom complexity.

Methods: All scorpion toxins belonging to the CSab scaf-fold were extracted from UNIPROT, aligned and analyzed fortheir phylogenetic association through Bayesian inference.This analysis was further used to assess position specific andpairwise analysis of selection within the toxin families.

Results and discussion: It was revealed that the func-tional redundancy of CSab toxins is the result of genetic




drift. Additionally, we showed a correlation betweenfunctional diversity of this scaffold in the venom and thepaleogeographic distribution of the scorpions. These find-ings present a paradigm shift to a model that requiresstrong directional selection pressure to induce structuraland functional recruitments, but allow the accumulation ofparalogs under neutral selection.

Conclusion: We suggest that functional redundancy ofvenoms is for a long-term evolutionary benefit rather thana consequence of the immediate–potency arms-race.

Keywords: venom evolution, genetic drift, functional redundancy10.1016/j.toxicon.2012.04.059

59. Understanding the Chemical Diversity of SpiderVenoms Using a Combined Genomic, Transcriptomicand Proteomic Approach

Sandy S. Pineda 1, Alun Jones 1, Graham M. Nicholson 2,Pierre Escoubas 3, John S. Mattick 4, Glenn F. King 1

1 Institute for Molecular Bioscience, The University of Queensland, Brisbane,Australia2 School of Medical & Molecular Biosciences, University of Technology,Sydney, Australia3VenomeTech, Valbonne, France4Garvan Institute, Sydney, AustraliaE-mail address: [email protected] (S.S. Pineda).

Background: Spiders and other venomous animalsdepend on the production of complex venom for defense,prey capture, and competitor deterrence. The major compo-nents of most spider venoms are disulfide-rich peptides ofmass 3–8 kDa. Recent mass spectrometric analyses indicatethat the venoms of some Australian funnel-web spiderscontain >500 different peptides. However, very little isknown about the genetic mechanisms used by these myga-lomorph spiders to generate such a complex chemicalcocktail.

Methods: In order to address this problem, we useda combined proteomic, transcriptomic, and genomicapproach to determine the complete range of peptidesexpressed in the venom of these spiders, including theunderlying architecture of the toxin-encoding genes, thenature of the mRNA transcripts, and the three-dimensionalstructure of the mature toxins.

Results: Proteomic analysis of the venomof an Australianfunnel-web spider (Hadronyche infensa) using both state-of-the-art OrbiTrap and LC-MALDI 4800 TOF-TOF mass spec-trometers yielded >2000 peptide masses. Two cDNAlibraries were constructed for analysis of the venom-glandtranscriptome: one from a single individual sequenced withSanger sequencing and a pooled library sequenced using 454technology. A two-step bioinformatic pipeline (ArachnoBaseand ToxSeek) was developed to analyse the >270,000 high-quality reads that were obtained. These analyses revealeda total of 32 superfamilies of venom peptides and proteins,including: (i) 25 disulfide-rich toxin families; (ii) 3 families ofenzymes; (iii) 1 family of cysteine-rich secretory proteins(CRISPs); and (iv) 3 families of secreted proteins. The numberof structural classes is currently under investigation, withselected toxins being expressed in E. coli with the aim ofsolving their 3D structure using NMR. Genomic analyses

have revealed that the disulfide-rich toxins are producedfrom intronless genes.

Conclusion: This work provides the most compre-hensive overview to date of the toxin landscape ofa prototypic mygalomorph spider venom. Neither of thetranscriptomic studies provided evidence for multi-toxintranscripts or for alternative splicing as a mechanism forgenerating toxin diversity. The results are consistent withthe one-gene:one-toxin paradigm proposed previously forvenomous cone snails. The holistic approach describedhere should enhance our understanding of how spidersand other venomous animals evolved such complexvenoms.

Keywords: spider venom, venom proteome, venom evolution, proteomics,transcriptomics, genomics, bioinformatics10.1016/j.toxicon.2012.04.060

60. Glycan Structures and Intrageneric Variations ofAcidic Phospholipases A2 from Tropidolaemus Venom

Inn-Ho Tsai 1,2, Hui-Ching Chang 2, Jin-Mei Chen 1,An-Chun Cheng 1, Kay-Hooi Khoo 1,2

1 Institute of Biological Chemistry, Academia Sinica, Taipei, Taiwan2 Institute of Biochemical Sciences, National Taiwan University, Taipei,TaiwanE-mail address: [email protected] (I.-H. Tsai).

Background: Pitvipers belonging to the Tropidolaemusgenus are endemic to southeastern Asia, and it may consistof four species. Tropidolaemus venoms are unique in thatthey contain special neurotoxins known as waglerins andonly a very little amount of hemorrhagins. However, theother venom components are not well understood.

Results: Phospholipases A2 (PLA2s) were purified andcharacterized using the venoms obtained from twodifferent regions, Sumantra and Sulawesi. An active PLA2could be isolated from both the samples with an approx-imate yield of 4w7% (w/w). Mass analyses and N-terminalsequencings revealed that the PLA2s in both the sampleswere different although their waglerins were identical.The samples probably derived from two species, namelyT. wagleri and T. subannulatus. This is consistent with therecent taxonomic and geographic study of this genus. TheN-terminal sequences of both the PLA2s were homologousto the acidic PLA2s of the other pitviper venoms whichcontain a conserved Glu6 residue. Interestingly, bothPLA2s appeared to be glycosylated at Asn14. Hydrolysis byPNGase F reduced their apparent masses from 16 to 14kDa. The released glycans were analyzed by MALDI-TOFand were found to be complex type oligosaccharideswithout sialylation. The acidic PLA2 from the Sumantrasample induced the aggregation of mouse platelets, whilethe PLA2 from Sulawesi inhibited the aggregation ofmouse platelets induced by ADP and collagen. Further-more, enzymatic removal of glycans from the PLA2s didnot significantly alter their effects on lipid hydrolysis andplatelet aggregation.

Conclusion: Tropidolaemus venoms demonstrate inter-esting novelty and biodiversity. This is the first report ofglycosylated snake venom PLA2s whose glycan structureshave been solved. The presence of glycans on these enzymes




warrants further analyses whichmay provide useful insightsinto the functional regulation of these molecules.

Keywords: Tropedolaemus venom, antiplatelet phospholipases, massprofile of N-glycans10.1016/j.toxicon.2012.04.061

61. A Phylogenetic Framework for the Study ofConvergence and Divergence in Scorpion Venoms

Ricardo C. Rodríguez de la Vega 1,2, Nicolas Vidal 21 Laboratoire Ecologie, Systématique et Evolution, Université Paris-Sud, OrsayFrance2Département Systématique et Evolution, Muséum National d'HistoireNaturelle, Paris FranceE-mail address: [email protected] (R.C. Rodríguezde la Vega).

Background: Mining on scorpion venoms using highthroughput techniques has revealed a large prevalence ofnon canonical components in scorpionvenoms,which in factoutnumber the typical toxin class. The evolutionary historiesof these scorpion venom components show puzzlingpatterns, where both extensive divergence and recurrentrecruitment are thought to have played important roles.

Methods: Non-redundant datasets were built for fiveprotein families found in scorpion venoms from at leastthree different taxonomic families. Non-venom orthologswere identified by database searching and tested as proxiesfor rooting venom proteins phylogenetic trees. MaximumLikelihood and Bayesian trees for each protein family werereconstructed and compared to a draft species tree basedon public mitochondrial sequences.

Results: High confidence rooted trees were obtained forthree venom protein families. In the remaining two families,non-venom orthologs were found nested within venomclades, thus suggesting independent recruitment events.

Discussion: Mapping of venom proteins trees onto thespecies tree reveals a tangled pattern of continuous recruit-ment and lineage-specific diversification. Reconstructing theevolutionary history of venom proteins remains a difficulttask due to multiple confounding factors. The use of a robustspecies phylogeny would certainly aid to resolve thediscrepancies found in thedifferentgene treesandhelp in thedistinction between convergent recruitment and divergence.

Conclusions: Inasmuch genomes can be regarded as thehistorical records of previous selection processes, themolecules making up the venoms should reflect the historyof the interspecies interactions that the producing lineageshave faced. It follows that reconstruction of their evolu-tionary histories should 1) clarify how venoms haveattained their remarkable complexity and 2) shed light overthe multi-organismic processes that have shaped theevolution of venomous organisms in the context of theirecosystemic networks.

Acknowledgments:The research leading to these results has received funding

from the European Union Seventh Framework Programme(FP7/2007-2013) under grant agreement No. 246556. RCRVstaying at the MNHN was funded by the Marie de Paris.

Keywords: convergence, divergence, evolution, scorpion, toxin, venom10.1016/j.toxicon.2012.04.062

62. Tentacles of Venom: Molecular Evolution of ColeoidVenoms

Bryan G. Fry 1, Tim Ruder 1, Dessi N. Georgieva 2,David Morgenstern 3, Glenn King 3, Eivind A.B. Undheim 1,3

1Venom Evolution Laboratory, School of Biological Sciences, The University ofQueensland, St. Lucia, Australia2 Laboratory of Structural Biology of Infection and Inflammation, Institute ofBiochemistry and Molecular Biology, University of Hamburg, Germany3Division of Chemistry and Structural Biology, Institute for MolecularBioscience, The University of Queensland, St. Lucia, AustraliaE-mail address: [email protected] (B.G. Fry).

Background: New insights into the evolution of venomsystems and the importance of the associated toxins cannotbe advanced without recognition of the true biochemical,ecological, morphological and pharmacological diversity ofvenom systems. A major limitation of the use of venomproteins has been the very narrow taxonomical rangestudied. Entire groups of venomous animals remain virtu-ally completely unstudied. One such group are the coleoids(cuttlefish, octopuses, and squid), which have only beenrecently revealed by us to share a common venomousancestor.

Methods: Eleven cuttlefish, octopuses, and squid specieswerefieldcollected fromtropical throughtopolarwaters, thusproviding wide taxonomical and ecological coverage. Venommolecular evolution was analysed using a combined proteo-mics/transcriptomics approach including mass spectrometry,2-gels, and cDNA libraries. The comparison of proteomic andtranscriptomic data allowed for rapid identification ofpeptide/protein types present in the venoms and subsequentdetermination of full-length transcript sequences.

Results: The combined approach revealed not onlyprotein/peptide types convergently recruited into the chem-ical arsenals of other venomous animals but also discoverednovel molecular scaffolds unique to coleoid venoms. Ourbioactivity studies also revealed unique temperature specificadaptations of enzymes found in the Antarctic species.

Discussion: Coleoid venoms were revealed to be ascomplex as other venoms that have traditionally been therecipient of the bulk of research efforts. The presence ofmultiple peptide/protein types convergently present otheranimal venoms reveals new information as to what char-acteristics make a peptide/protein type amenable forrecruitment into chemical arsenals.

Conclusion: Coleoid venoms have significant potentialnot only for understanding fundamental aspects of venomevolution but also as an untapped source of novel toxins foruse in drug design and discovery.

Keywords: coleoid, venom, venomics, proteomics, transcriptomics, toxinevolution10.1016/j.toxicon.2012.04.063

63. Discovery of the Nicotinic Receptor ToxinAnabaseine in a Polystyliferan Nemertine

William R. Kem1, Juan Junoy 2

1University of Florida College of Medicine, Dept Pharmacology andTherapeutics, Gainesville, FL, USA2Departamento de Biologia Animal, Universidad de Alcala, Alcala deHenares, SpainE-mail address: [email protected] (W.R. Kem).





Background: Nemertines, a phylum of predominantlymarine worms, prey on other animals and defend them-selves with toxins. The anoplan nemertines lack a proboscisstylet for puncturing their prey and secrete neurotoxic andcytotoxic peptides. The hoplonemertines are enoplan(armed) worms that have a proboscis armedwith onemorestylets. Hoplonemertines are systematically divided intothe relatively diverse order Monostylifera that is relativelycommon in certain benthic zones and the less readilycollected order Polystylifera that are bathypelagic as well asbenthic in distribution. Monostyliferan nemertines areknown to produce alkaloidal toxins (including anabaseine)that affect nicotinic acetylcholine receptors and pyridinechemoreceptors (Bacq, 1936; Kem, 1971; Kem and Soti,2001; Kem et al., 2006). Anabaseine has been a leadcompound in the design of alpha7 nicotinic receptoragonists to treat disorders of cognition such as Alzheimer'sdisease and schizophrenia (Kem et al., 2004; Freedman etal., 2008).

Methods: We recently obtained two live specimens ofthe sublittoral benthic polystyliferan Paradrepanophoroscrassus (PC) from the northwest coast of Spain. Bacq (1936)found this species to contain a substance(s) acting likenicotine on autonomic ganglia. We used Ehrlich's reagent(Kem et al., 1971) for anabaseine detection and HPLC andmass spectrometric methods for isolation and identifica-tion of anabaseine and related alkaloids. Ethanolic preser-vative was used to extract the toxins.

Results: We have demonstrated that Pc containscontains high concentrations of anabaseine in its bodyproper and proboscis but lacks 2,3'-bipyridyl, nemertellineand other alkaloids found in the benthic monostyliferanAmphiporus angulatus.

Conclusions: Our data indicate that the biosyntheticmachinery for producing anabaseine probably wasacquired by a common ancestral hoplonemertine beforethe evolutionary divergence of these two hoplonemertineorders.

Keywords: anabaseine, nicotinic, nemertine10.1016/j.toxicon.2012.04.064

64. Centipede Venoms: Old and Unusual

Eivind A.B. Undheim 1,2, Alun Jones 1, John W. Holland 1,Rodrigo A.V. Morales 3, Brit Winnen 1, Bryan G. Fry 2,Glenn F. King 1

1Division of Chemistry and Structural Biology, Institute for MolecularBioscience, The University of Queensland, St. Lucia, Australia2Venom Evolution Laboratory, School of Biological Sciences, The University ofQueensland, St. Lucia, Australia3Monash Institute of Pharmaceutical Sciences, Parkville VIC, AustraliaE-mail address: [email protected] (E.A.B. Undheim).

Background: At 420 million years old centipedesrepresent one of the oldest extant arthropod venomsystems. However, despite the ancientness of thesevenoms, almost nothing is known about their componentsor molecular evolution.

Methods: Five centipede species were selected toprovide both wide taxonomic coverage and a phylogenetictimeline to date evolutionary events. Emphasising the

Scolopendridae due to their large size, availability, andclinical importance, the following species were selected torepresent over 400 million years of evolution and to allowfor comparisons at the order, subfamily, genus, and specieslevel: Thereuopoda sp., Ethmostigmus rubripes, Cormoce-phalus westwoodii, Scolopendra alternans, and Scolopendramorsitans. Milked venoms were analysed using a combinedproteomics/transcriptomics approach including massspectrometry, 2-gels, and cDNA libraries. The comparisonof proteomic and transcriptomic data allowed for rapidsequence determination and, crucially, identification ofposttranslational modifications.

Results: We provide the first comprehensive insightinto the chilopod “venome”, revealing novel scaffoldsunique to centipede venoms as well as scaffold types con-vergently recruited into other venoms. We also presentmultifunctional transcripts and transcripts with tandemlyrepeated sequences, which are unusual to invertebratevenoms.

Discussion: Centipede venoms appear to differsubstantially from the venoms of other arthropods in theabundance of high molecular weight components. Theancient evolutionary history of centipedes is also apparentfrom the differences at the highest taxonomic level, whichdiverged about 400 mya.

Conclusion: The presence of a wide range of novelproteins and peptides in centipede venoms highlightsthese animals as a rich source of novel bioactive molecules.Understanding the evolutionary processes behind theseancient venom systems may not only aid in directing bio-prospecting efforts, but it will also broaden our under-standing of which traits make proteins and peptidesamenable to neofunctionalisation.

Keywords: centipede, venom, venomics, proteomics, transcriptomics,toxin evolution10.1016/j.toxicon.2012.04.065

65. The Phylogenetic Scale of Venom Variation inHaplogyne Spiders

Greta J. Binford, Miles Dale, Andrew Wood, Jared Delahaye,Ian Voorhees, Jennifer Mullins, Pamela A. Zobel-ThroppLewis & Clark College, Dept. Biology, Portland, OR, USAE-mail address: [email protected] (G.J. Binford).

Background: Haplogynes are a higher-level clade ofaraneomorph spiders that includes many taxa of interestwith respect to their venom composition. Some notablehaplogynes include pholcids (“daddy long legs” or cellarspiders), plectreurids, and families in the scytodoid super-family such as spitting spiders (Scytodidae) and the sicariidfamily that includes brown recluse (Loxosceles) and six-eyed sand spiders (Sicarius).

Methods: With a goal of analyzing the phylogeneticscale of venom variation in spiders in general, we arecomparing venom gland transcriptomes and proteomesfrom representatives of this group selected based on theirphylogenetic position. Our data include some comparisonsamong relatively closely related taxa (common ancestorwithin the last 30 million years) and among other more




distant relatives (common ancestor roughly 200 millionyears old). Our center of focus is comparisons amonglineages of sicariids, that been evolving for over 100millionyears in the context of being generalist, ground-dwellingpredators of arthropods. We compare sets of sicariid toxinswith toxins from scytodidae, pholcidae and non-haplogynearchaeid spiders.

Results: We have discovered venom peptide toxinfamilies that appear to be distinct for sicariidae, andothers that are expressed across haplogynes. While sometoxins are phylogenetically widespread, there are strikingdifferences among lineages in relative abundance of thesetoxins. We will discuss patterns of positive selectionwithin some of the most common and widespead toxinlineages. Together these data help illuminate the evolu-tionary dynamics of venom functional complexes inspiders.

Keywords: diversity, evolution, phylogeny10.1016/j.toxicon.2012.04.066

66. b-defensin-like genes in Brazilian Poisonous Snakes

Poliana G. Corrêa 1, Taís Machado 1, Valdir J. Germano 2,Daniela P.T. Gennari 2, Álvaro R.B. Prieto-da-Silva 3,Nancy Oguiura 1

1 Instituto Butantan, Laboratório Especial de Ecologia e Evolução, São Paulo,Brazil2 Instituto Butantan, Laboratório de Herpetologia, São Paulo, Brazil3 Instituto Butantan, Laboratório de Genética, São Paulo, BrazilE-mail address: [email protected] (P.G. Corrêa).

Background: b-Defensins are antimicrobial peptides(AMP) found in vertebrates. They constitute an importantand conserved component of innate immunity with anti-microbial activities. These molecules are also reported inthe venoms of sea anemones, snakes and platypus. InCrotalus durissus terrificus, it is known two peptides with b-defensin scaffold: crotamine, a small basic myotoxin ofrattlesnake venom, and crotasin, a gene expressed abun-dantly in several rattlesnake tissues, but scarcely in thevenom gland. In this study we prospected b-defensin-likegenes in snakes of Viperidae family by PCR.

Methods: Genomic DNA from different species ofBothrops, Bothropoides, Rhinocerophis and Lachesis snakeswere used as template. The primers were designed basedon the signal peptide (SP) and 3’UTR, conservedsequences from crotamine and crotasin genes. Theamplifyed sequences were aligned with CodonCodeAligner and the phylogeny analyzed by maximum parsi-mony using TNT1.1.

Results: From nine Viperidae snakes we obtained tendifferent sequences. The genes presented 3 exons and 2introns that code the SP and the mature b-defensin. Thesize of first intron vary greatly (0.4 - 1.7kb) whereas thesecond was conserved (w153 bp). The mature peptides(MP) have the six cysteines (conserved in the b-defensinfamily), basic amino acids residues clustered in the carboxiregion, net charge fromþ2 toþ12, a N-terminal glutamine,a conserved glycine at ninth position and a C-terminallysine.

Discussion: The gene organization is similar to crot-amine, crotasin and other genes found in lizard andteleost fish. The SP and introns sequences are moreconserved than MP sequences. The Ka/Ks ratio suggestedan accelerated evolution on exon 2. Phylogenetic analysesusing introns or exons did not recover the phylogeneticrelationships obtained with mitochondrial DNA forViperidae species, probably due to differential selectionpressure of these two types of data. Between thesenuclear sequences, the intron phylogeny has a betterresolution, recovering individuals of the same species assister groups.

Conclusions: The gene structure of b-defensin in snakesis different from that found in mammals and birds, but issimilar to that found in another reptile, the green lizardAnolis carolinensis. As expected to this family of genes, itwas observed an accelerated evolution on exon 2, whatincrease the peptide variability and enable the animal toprotect against diverse pathogens.

Financial Support: FAPESP and INCTTox.

Keywords: rattlesnakes, b-defensins, gene structure, toxin evolution10.1016/j.toxicon.2012.04.067

67. Comparative Analysis of Transcriptomes ofPhoneutria pertyi and P. nigriventer Venom Glands

Marcelo R.V. Diniz 1, Camilla R.L. Machado 1, B. Paiva AnaLuiza 1

1Centro de Pesquisa e Desenvolvimento Carlos Ribeiro Diniz, FundaçãoEzequiel Dias, Belo Horizonte, BrazilE-mail address: [email protected] (M.R.V. Diniz).

Background: Species of the genus Phoneutria knownas "aranha-armadeira" or armed spiders are responsiblefor a large number of spider bites in Brazil. Until recently,all studies on the venom of the genus have been restrictedto the species P. nigriventer. However, some recent pro-teomic studies revealed that other species venoms alsocontains a wide variety of proteins and peptides,including neurotoxins which act on the ion channels andchemical receptors of the neuro-muscular systems ofinsects and mammals. Thus, these venoms have emergedas invaluable tools for research, drug discovery and drugdevelopment with application in medicine andagriculture.

Methods: In order to find novel venom componentswith biological activity and to provide a database forcomparative study with the previously described P. nig-riventer venom gland transcriptome, we constructeda plasmidial cDNA library from the species Phoneutria pertyimRNA venom gland to generate Expressed Sequence Tags(ESTs) data.

Results: After editing, 710 good quality reads wereclustered and 295 unique sequences were obtained (106contigs and 189 singlets). Of these, 197 (67%) had a highdegree of homology to spiders toxins deposited in theUniprot database, most are P.nigriventer toxins isoforms.We observed that P. pertyi venomgland transcriptomeweremore abundant in insecticidal toxin sequences thanP. nigriventer one. We also found new sequences for




putative toxins in P. pertyi transcriptome, indicating thatthey can be novel toxins.

Conclusions: These results show that although thesespider venoms contain a similar range of toxins iso-forms, the expression levels of each type of toxins aredifferent, and also they contain toxins with uniquesequences, what can suggest adaptation to differentenvironments.

Keywords: Phoneutria venom gland, transcriptome, neurotoxin10.1016/j.toxicon.2012.04.068

68. Substrate range and specificity of the SicToxphospholipase D toxins

Daniel M. Lajoie 1, Greta J. Binford 2, Vahe Bandarian 1,Matthew H.J. Cordes 1

1Department of Chemistry and Biochemistry, University of Arizona, Tucson,AZ, USA2Department of Biology, Lewis and Clark College, Portland, OR, USAE-mail address: [email protected] (D.M. Lajoie).

Background: Certain phospholipase D (PLD) toxinsfrom the venoms of the Sicariidae spider family can inducenecrotic lesions in mammalian tissue. The gene family(‘SicTox’) containing these PLD toxins is split into two wellresolved clades: alpha and beta. The alpha-clade membersgenerally exhibit enzymatic activity towards the substratesphingomyelin (SM); in contrast, the beta-clade toxinsexhibit diminished PLD activity towards SM. We arestudying the catalytic activities of SicTox family PLDenzymes to gain insights into the evolution of substratespecificity and eventually correlate them with the biolog-ical function of these proteins as toxins.

Methods: We have cloned and can heterologouslyexpress a representative member from both the alpha- andthe beta- clades. The beta-clade toxin is from the Sicariidaespecies Sicarius terrosus. The alpha-clade member is fromLoxosceles arizonica, whose venom is known to be signifi-cantly harmful to humans. We are utilizing 31P-NMRspectroscopy and spectrofluorometric assays to screen forphospholipase activity against a broad range of substrates.

Results: The alpha-clade enzyme is multispecific andexhibits PLD activity towards sphingomyelin and lyso-phosphatidylcholine. In contrast, the beta-clade toxin hasdiminished PLD activity for either of the choline-con-taining substrates. This is the first time a SicTox geneproduct has been biochemically characterized froma Sicarius species.

Conclusion: We can heterologous express and purifyactive SicTox enzymes. 31P-NMR spectroscopy and enzymecoupled spectrofluorometric assays allows us to screena large variety of phospholipid compounds for phospho-lipase activity to compare their catalytic efficiencies. Theresults to date indicate a difference in the substrateprofiles of the proteins from the alpha- and beta- cladePLD toxins.

Keywords: SicTox, phospholipase D, Sicariidae10.1016/j.toxicon.2012.04.069

D. Hemostasis

69. Recurrent, Persistent, or Late, New-OnsetHematologic Abnormalities in CrotalineSnakebite

Steven A. Seifert 1,2, Ronald I. Kirschner 3,Nancy Martin 1

1New Mexico Poison and Drug Information Center, Albuquerque,NM, USA2 School of Medicine, University of New Mexico, Albuquerque,NM, USA3Department of Emergency Medicine, University of NebraskaMedical Center; Nebraska Regional Poison Center, Omaha,NE, USAE-mail address: [email protected] (S.A. Seifert).

Background: Hematologic effects from rattlesnakeenvenomation exhibit a phenomenon of recurrent, persis-tent or late, new onset (late) abnormalities in some Fabantivenom-treated patients 4 or more days post-enveno-mation. Indicators that reliably identify or exclude thosepatients at risk of late hematologic effects have not beendeveloped.

Methods: This was a retrospective, observational caseseries of rattlesnake bite records at two US poison centers.The sensitivity, specificity, positive predictive value (PPV)and negative predictive value (NPV) for D-dimer, fibrin-ogen, platelets, platelet count trend, INR and PTT associ-ated with late hematologic abnormalities, weredetermined.

Results: Three hundred seventy six cases werereviewed. Sixty cases met inclusion criteria. Overall, 17 of60 patients (28%) had a hematologic abnormality asa result of envenomation. Eleven of 60 patients (65% ofthose with a hematologic abnormality; 18% overall)developed late hematologic abnormalities 4 or more dayspost-envenomation. Four patients had late, new onsethypofibrinogenemia and/or thrombocytopenia. All wereassociated with early D-dimer elevation and/or plateletrise in response to FabAV treatment, respectively. Normalhematologic parameters in the first 48 h post-envenoma-tion and the lack of a greater than 20% rise in plateletswithin 4 h post-antivenom administration had a 100% NPVfor late hematologic effects.

Conclusions: Patients with early onset hypofi-brinogenemia, a positive D-dimer, thrombocytopenia, ora 20% increase in platelet count within 4 h post-treat-ment had significant likelihood of late hematologiceffects. Patients in whom fibrinogen, D-dimer, INR, PTT,and platelet counts remained normal throughout the first48 h post-envenomation, and who did not exhibit a >

20% increase in platelet count within 4 h post-antivenomadministration, did not develop late hematologic effects.This work, if validated, may help to identify thosepatients at particular risk of hematologic recurrence;those with risk of new, late onset hematologic effects;and those in whom late hematologic effects are unlikelyto occur.



Table 4Cases with recurrent, persistent or late, new onset increased INR or PTT

Case # HighestearlyPT/INR

LowestFibrinogen

D-dimer Highest4þ daysPT/INR

Any LateAbnormality

3 1.9 (INR) < 50 Positive 8.8 (INR) R3 24 (PTT) < 50 Positive 120 (PTT) L4 1.5 (INR) 83 ND 1.4 (INR) P5 1.3 (INR) 121 ND 1.4 (INR) R11 41 (PTT) 167 Positive 43 (PTT) R

Units: Fibrinogen ¼ mg/dL; INR ¼ samples/normal; PTT ¼ secondsAbbreviations: International normalized ratio (INR); Partial thromboplastintime (PTT); R¼Recurrent; P¼Persistent; L¼Late, new onset; ND¼Not done.

Table 1Sensitivitey, Specificity, Positive and Negative Predictive Values compared between the clinical trials, the published data of Ruha, et al, and this study.

Current Study (n ¼ 60) Clinical Trials^ (n ¼ 42) Ruha et al.* (n ¼ 66)

Pre-48 hour Parameter Sens1 Spec1 PPV1 NPV1 Sens Spec PPV NPV Sens Spec PPV NPVFibrinogen 60% 89% 43% 94% 67% 59% 76% 76% 67% 78% 43% 94%D-dimer 100% 56% 21% 100% 87% 69% 65% 89%Fibrinogen wnl1 & D-dimer wnl - 100% - 100%Fibrinogen wnl & D-dimer increased 100% - 14% -Platelet Count 75% 81% 43% 95% 78% 83% 58% 93% 75% 85% 43% 96%Platelet Trend 100% 76% 43% 100%Platelets wnl & Trend < 20% increase - 100% - 100%Platelets wnl & Trend > 20% increase 100% - 25% -INR1 100% 63% 14% 100% 71% 68% 33% 91%PTT1 50% 85% 20% 96% 40% 85% 29% 90%ANY ABNORMAL 72% - 28% - 100% - 76% - 100% 43%ALL WNLx - 100% - 100% 100% 63% - 100% - 88%

Abbreviations: 1Sensitivity (Sens), specificity (Spec), Positive Predictive Value (PPV) and Negative Predictive Value (NPV), Within normal limits (wnl),International normalized ratio (INR), Partial thromboplastin time (PTT)^In the clinical trials, FSPs, not D-dimers, were measured and the data presented is for FSPs.1

*In Ruha's study, fibrinogen was combined (and/or) with PT/INR and fibrinogen data is for the combined analysis.5

x The clinical trials did not include abnormal FSPs in their definition of hematologic abnormality and did not include platelet trend. The study by Ruha, et aldid not include D-dimer or platelet trend.

Table 2Cases with recurrent, persistent or late, new onset thrombocytopenia.

Case # Lowestearlyplatelets

Plateletsw/in 4 h

>20%plateletincrease

Lowest4þ daysplatelets

LateThrombocytopenia

8 25 146 Y 85 R10 25 336 Y 61 R5 59 237 Y 36 R3 95 230 Y 102 R4 47 ND ND 138 R23 112 ND ND 56 P2 154 243 Y 47 L6 196 247 Y 91 L

Units: Platelets ¼ x1000/uL.Abbreviations: R¼Recurrent; P¼Persistent; L¼Late, new onset; Y¼Yes;ND¼Not Done.

Table 3Cases with recurrent, persistent or late, new onset (late)hypofibrinogenemia.

Case # LowestearlyFibrinogen

D-dimer Lowest4þ daysFibrinogen

Latehypofibrinogenemia

3 <50 Positive <50 R9 133 Positive 54 R4 83 ND <50 P7 252 Positive 134 L5 272 Positive 121 L

Units: Fibrinogen ¼ mg/dL.Abbreviations:R¼Recurrent;P¼Persistent; L¼Late,newonset;ND¼Notdone.


Previously published: Clinical Toxicology (2011); 49:324–329.

Keywords: Crotalinae, hematologic effects, recurrence10.1016/j.toxicon.2012.04.070

70. Purification and Characterization of New PlateletAggregation Inhibitor with Dissociative Effect onADP-Induced Platelet Aggregation, from ProtobothropsVenom in Japan

Etsuko Oyama, Naomichi Furudate, Kotaro Senuki,Hidenobu TakahashiMeiji Pharmaceutical University, Department of Hygienic Chemistry, Tokyo,JapanE-mail address: [email protected] (E. Oyama).

Background: Snake venom disintegrins contain the RGDor KGD sequence in a homologous position, and inhibit theinteraction between fibrinogen and the integrin aIIbb3. Theclass P-III snake venom metalloproteinases (SVMPs) containdisintegrin-like, and cysteine-rich domains, and also inhibitplatelet aggregation. Recent work has shown interactionbetween platelet integrins and disintegrin or cysteine-richdomains of P-III SVMP such as jararhajin [1] from BothropsjararacavenomandatrolysinA [2] fromCrotalus atroxvenom.In this study, we report the purification and characterizationof new inhibitors,with adissociative reactionofADP-inducedplatelet aggregation, from Protobothrops venom.

Methods: Proteins were purified by gel-filtration andion-exchange chromatography. Platelet aggregation testsfollowed the turbidimetric method of Born [3]. The bindingassay of fibrinogen and aIIbb3 was performed by ELISA.

Results and Discussion: We purified three inhibitors,named as SV-PAD-1 (from P. elegans venom; [4]), PT-PAD(from P. tokarensis venom), PF-PAD (from P. flavoviridisvenom). Three proteins showed a single protein band andthese molecular weights were about 110 kDa on SDS-PAGEunder reducing conditions. We suggest that these proteinsare P-III SVMP because of these proteins showed enzymaticactivity and those activities were completely inhibited by



EDTA. SV-PAD-1, PT-PAD, and PF-PAD strongly inhibitedADP-induced platelet aggregation. IC50 values of SV-PAD-1,PT-PAD, and PF-PAD were about 20-53 nM, and that of ele-gantin (from P. elegansvenom; [5]) a disintegrin,was300nM.Furthermore, three inhibitors promptly dissociated plateletaggregation in rabbit platelet-rich plasma stimulated with 2mM ADP. 50% dissociative concentrations of SV-PAD-1 andPT-PAD on ADP-induced platelet aggregation were 16 nMand 12 nM, however elegantin did not dissociate on plateletaggregation even at 1 mM. In the binding assay betweenfibrinogen and aIIbb3, SV-PAD-1 and PT-PAD hardly inhibi-ted fibrinogen binding to aIIbb3, whereas only PF-PADinhibited binding between aIIbb3 and fibrinogen.

Conclusions: SV-PAD-1, PT-PAD, and PF-PAD are uniqueproteins from snake venoms that promptly dissociated ADP-induced platelet aggregation. We suggest that the mecha-nism(s) of actionof these three inhibitors is different from thatof elegantin and these inhibitors will be useful for the inves-tigation of the mechanism of action of platelet dissociation.

Fig. 1. Dissociation of SV-PAD-1 and elegantin on ADP-induced plateletaggregation. The experimental procedures and conditions were described inMethods.

References

Lambert L. J. et al., (2008) J. Biol. Chem. 283, 16665-16672.Jia L. G. et al., (2000) Arc. Biochem. Biophy. 373, 281-286.Born G. V., (1962) Nature 194, 927-929.Oyama E. et al., (2009) Toxicon 53, 706-712.Scaloni A. et al., (1996) Biochem. J. 319, 775-782.

Keywords: protobothrops venom, platelet aggregation, dissociation,purification10.1016/j.toxicon.2012.04.071

71. Antiplatelet Activity and Mass Spectrometric Studyof Venoms from Two Iranian Vipers, Echis carinatus andCerastes persicus fieldi

Toktam Mehdizadeh Kashani 1, Hossein Vatanpour 1,Hossein Zolfagharian 2, Hassan Hooshdar Tehrani 3,Farzad Kobarfard 3,4

1Department of Toxicology, School of Pharmacy, Shahid Beheshti Universityof Medical Sciences, Tehran, Iran

2Department of Venomous Animals and Antivenom Production, Razi Vaccineand Serum Research Institute, Karaj, Iran3Department of Medicinal Chemistry, School of Pharmacy, Shahid BeheshtiUniversity of Medical Sciences, Tehran, Iran4 Phytochemistry Research Center, Shahid Beheshti University of MedicalSciences, Tehran, IranE-mail address: [email protected] (T.M. Kashani).

Background: Platelet aggregation inhibitory effect andanticoagulant properties of fractions separated from thevenoms of Cerastes persicus fieldi and Echis carinatus wereinvestigated.

Methods: The partial fractionation was performed ona Sephadex G-100 column.

Results: Two fractions separated from Cerastes persicusfieldi showed antiplatelet aggregation activity on ADP (200mM)-induced platelet aggregation (ca 80% inhibition). Themolecular mass of the most potent platelet aggregationinhibitor from Iranian Cerastes persicus fieldi venom wasdetermined to be 4462 Da by LC-ESI-MS. Mass spectrom-etry of the whole venom of Iranian Cerastes persicus fieldiresulted in two major components with molecular massesof 564 and 1158 Da. Attempts to measure the antiplateletaggregation activity of crude Echis carinatus venom and itsfractions were not successful due to the protein coagulationof the plasma samples after addition of the venom. LC-MSstudy of the whole venom from Iranian Echis carinatuspermitted us to detect a componentwithmolecular mass of13 kDa. Anticoagulant activities of the venoms were alsoevaluated. Total venom of Echis carinatus showed antico-agulant activity in PT test, while its fractions showed pro-coagulant activity.

Keywords: anticoagulant, antiplatelet aggregation, snake venom, Echiscarinatus, Cerastes persicus fieldi, gel filtration, LC-ESI-MS10.1016/j.toxicon.2012.04.072

72. A Novel Family of Factor Xa Inhibitors from theSalivary Gland of Sandfly Vector of Leishmaniasis,Lutzomyia longipalpis.

Nicholas Collin 1, Teresa Assumpcao 1, Daniella Mizzurini 2,Robson Monteiro 2, Jesus Valenzuela 1, Ivo Francischetti 11 Laboratory of Malaria and Vector Research, NIAID/NIH, Bethesda, MD, USA2 Institut od Medical Biochemistry, Federal University of Rio de Janeiro, BrazilE-mail address: [email protected] (I. Francischetti).

Background: Salivary glands from blood suckingarthropods display a notable repertoire of anti-hemo-statics, including vasodilators, platelet and coagulationinhibitors. Several distinct families of anticoagulantstargeting FXa have been described, including Kunitzinhibitors, serpins, antistasin-like inhibitors, amongothers.

Results: We discovered that the main anticoagulant ofthe sandflies, L. longipalpis, and P. papatasi, is part ofa novel family of FXa inhibitors, herein named Lufaxin,which does not display sequence similarity to any anti-coagulant discovered so far, or protein deposited in thedatabase. Lufaxin is a 34 kDa protein with 8 disulphidebridges, and was found to be a slow, tight, reversible and




specific inhibitor of FXa. Lufaxin blocks FXa catalyticactivity and prothrombinase assembly, but does notinhibit thrombin, trypsin, FIXa, FXIIIa, tryptase, plasmin,FX or DEGR-FXa (catalytic site inhibited FXa). SPRrevealed that Lufaxin interacts with FXa with high affinity(kDw 5 nM), while analytical experiments demonstratedthat the interaction is stoichiometric. Notably, Lufaxinalso prevents PAR2 activation in glioblastoma cell linesactivated with FXa, and prevents paw edema formationtriggered by FXa injection. Finally, Lufaxin potentlyinhibits thrombus formation in vivo (triggered by chloricferric), promotes bleeding, and prolongs PT and aPTT exvivo.

Discussion: Lufaxin is a novel family of FXa inhibitors,which displays both antiinflammatory and antithromboticactivity.

Conclusions: Its unique sequence represents a noveltool to understand the structure of FXa, or is a prototype todevelop novel anticoagulants targeting FXa.

Keywords: Factor Xa, anticoagulants, blood sucking10.1016/j.toxicon.2012.04.073

73. Novel Anticoagulants from Snake Venom

V.M. Girish 1, R. Manjunatha Kini 1,21Department of Biological Sciences, Faculty of Science, National University ofSingapore, Singapore2Department of Biochemistry, Medical College of Virginia, VirginiaCommonwealth University, Richmond, Virginia, USAE-mail address: [email protected] (R.M. Kini).

Review: Anticoagulants prevent the formation ofunwanted blood clots that can lead to heart attack or strokeand that result in a large number of deaths in developedcountries. Currently available drugs have some drawbacks,including their non-specific actions. Therefore, novel anti-coagulants that target specific steps in the coagulationpathway are being sought. Recently, we have isolated andcharacterized several anticoagulants from various snakevenoms. These anticoagulants belong to the three-fingertoxin family and exert their effects on the extrinsic pathwayof the blood coagulation system. Despite similarity inoverall three-dimensional structure, three-finger toxinsrepresent an interesting family of anticoagulants as theyare able to recognize various distinct targets in the bloodcoagulation system. Some of these toxins target theextrinsic tenase complex, whereas others target the pro-thrombinase complex. Further, they all exhibit their anti-coagulant functions through distinct mechanisms. Here, wewill describe the functional characterization of a few ofthese novel anticoagulants. We will also discuss themechanisms of their anticoagulant functions. Under-standing the structure-function relationships of theseanticoagulant toxins will help in designing potential leadsfor future anticoagulation therapies and hence are of greatinterest.

Keywords: snake venom, three-finger toxins, anticoagulants10.1016/j.toxicon.2012.04.074

74. Plant Latex Proteases: An Insight into theirProcoagulant Activity

H.V. Shivaprasad 3, M Yariswamy 1, R Rajesh. 3,B.S Vishwanath 1,2

1Department of Studies in Biochemistry, University of Mysore, India2Karnataka State Open University, Manasagangotri, Mysore, India3Department of Microbiology and Immunology, University of Maryland,Baltimore, USAE-mail address: [email protected] (H.V. Shivaprasad).

Background: Latex, a milky fluid characteristic ofcertain angiospermic plant families, has been widely usedfrom time immemorial as a hemostatic agent. There hasbeen a lack of sufficient scientific and biochemicalevidence on the mechanism of hemostasis and the presentstudy was to characterize the procoagulant nature of latexproteases.

Methods: Latex from plants belonging to Apoc-yanaceae, Asclepiadaceae and Euphorbiaceae families wereprocessed to get the protein-rich fraction, which was usedas the protease source. The proteolytic assay was carriedout using casein as substrate and the nature of proteaseswas substantiated using specific protease inhibitors. Theeffect of latex proteases on blood coagulation was deter-mined using citrated plasma from healthy volunteers andfrom Hemophilia patients. Thrombin-like and Plasminactivity of the crude and purified proteasewas determinedusing purified fibrinogen and washed plasma clotrespectively by spectrophotometric and electrophoreticmethods. Pergularain e I, a “thrombin-like” cysteineprotease from Pergularia extensa latex was purified tohomogeneity using cation exchange chromatography. Thehomogeneity andmolecular mass were determined by RP-HPLC and MALDI-TOF analyses. The cleavage site of Per-gularain e I was identified by MALDI-TOF analysis usingpurified fibrinogen.

>Results: The protein-rich fractions of different latexesshowed moderate to high proteolytic activity and hydro-lyzed casein in a dose-dependant manner. The proteaseactivity of Apocyanaceae and Euphorbiaceae was inhibitedby PMSF and Benzamidine, whereas, IAA inhibited Ascle-piadaceae latex proteases. Protease fraction from all thelatexes exhibited procoagulant activity and decreased theplasma recalcification time in a dose-dependant manner.Asclepiadaceae family cysteine proteases possessed“thrombin-like” activity, enabling the clotting of plasmawithout supplementation of Ca2þ. Cysteine proteases alsodecreased clotting time in plasma from hemophiliapatients. Pergualrain e I, a 23.356 K.Da basic glycoproteincontained about 20% carbohydrate. It cleaved fibrinogenbetween Arg and Gly, similar to thrombin.

Discussion: Proteases are abundant in plant latex and areresponsible for the pharmacological effects of the latex. Anygiven plant family contains only one type of protease. Thelatex cysteine proteases exhibit procoagulant activity byThrombin-like activity, whereas the mechanism of serineproteases is not clear. The clot inducing and dissolvingactivity of latex proteases is vital for their involvement inwound healing process. Pergularain e I acts similar tothrombin to release Fibrinopeptides A and B fromfibrinogen.




Conclusion: The detailed study on characterization andmolecular mechanisms of latex proteases will providebiochemical basis for the extensive use of latex as a hemo-static agent and will be of potential therapeutic use inthrombotic disorders.

Keywords: latex proteases, thrombin-like, hemophilia, fibrinopeptides10.1016/j.toxicon.2012.04.075

Fig. 1. ADP induced Platelet Aggregation process. BlueNegative control, BlackADP induced aggregation. Green ADP induced aggregation of 20 mg Lahirintreated blood. Red treated with higher concentration of Lahirin (40 mg).

75. The P-I Metalloproteinase from Cerastes cerastesSnake Venom Inhibits Human Platelet Aggregation

Hinda Boukhalfa-Abib 1,2, Fatima Laraba-Djebari 1,21 Laboratory of Cellular and Molecular Biology, Dept. of Cellular andMolecular Biology, Faculty of Biological Sciences, University of Science andTechnology Houari Boumediene, Algiers, Algeria2 Laboratory of Research and Development on Venom, Pasteur Institute ofAlgeria, Algiers, AlgeriaE-mail address: [email protected] (H. Boukhalfa-Abib).

Background: Snake venoms contain various metal-loproteases that are highly toxic, resulting in a severebleeding by interfering with the blood coagulation and bydegrading the basement membrane or extracellular matrix(ECM) components. It has been suggested that hemorrhagicmetalloproteases interact in a specific way with plateletsurface proteins resulting in an alteration of platelet func-tion. However, the exact mechanism of venom-inducedhemorrhage is not yet fully understood.

Methods: The P-I class SVMP, CcH1, was purified fromCerastes cerastes venom by a combination of gel filtration,ion exchange, affinity and RP- HPLC chromatography. ThisSVMP CcH1 is a 25 kDa weakly hemorrhagic metal-loproteinase causing a variety of local tissue damage. In thisstudy, we report the functional characteristics of CcH1 onplatelet aggregation and we evaluated the effect of CcH1 onextracellular matrix components (laminin and type IVcollagen).

Results: CcH1 was able to preferentially hydrolyze thea-chain of fibrinogen and fibrin. Proteolytic activity ofthe enzyme was completely inhibited by metal chelatingagents but not by other typical protease inhibitors. Thisenzyme principally degrades the laminin and type IVcollagen dose-dependent manner. It is interesting;however, that CcH1 strongly suppresses ADP-inducedhuman platelet aggregation in a dose-dependentmanner.

Discussion: The hemorrhagic mechanism of CcH1 couldbe summarized as follows. i), CcH1 is able to hydrolyzefibrinogen key molecule in blood coagulation and plateletaggregation. ii), this molecule has proteolytic activity onextracellular matrix proteins which are critical componentsin structure-function of the vascular basement. It ispossible to hypothesize that the metalloprotease domain ofCcH1 could contribute to the hemorrhagic activity of theenzyme. Such hypothesis can be strongly supported by thereports that the class P-III metalloproteases usually exhibitmore hemorrhagic activity than the class P-I metal-loprotease containing only a metalloprotease domain.

Conclusions: the P-I metalloproteinase CcH1 presentsa fibrinolytic activity and hydrolyze also laminin and

collagen IV. This weakly hemorrhagic proteinase inhibitsalso ADP-induced human platelet aggregation. This studycontributes to the understanding of the functional mech-anisms of metalloproteinases. Furthermore, the weakhemorrhagic of CcH1, could be used as a therapeutic tool inthe treatment and prevention of thrombotic diseases.

Keywords: snake venom, hemorrhagic metalloproteinase, plateletaggregation10.1016/j.toxicon.2012.04.076

76. An Antiplatelet Peptide, Lahirin, from IndianMonocled Cobra Venom

C. Chandra Sekhar, Dibakar ChakrabartyBITS Pilani K.K Biral Goa Campus, Department of Biological Sciences,Goa, IndiaE-mail address: [email protected] (C. Chandra Sekhar).

Background: A low Molecular weight (6.5 kDa) fibrino-genolytic peptide has been purified from the Indian mono-cled cobra (Naja kaouthia) venom and was named Lahirin. Itis a a-fibrinogenasewhich cleaves the Aa chain of fibrinogenin a dose and time dependentmanner. It also dissolved fibrinclots developed in vitro. Lahirin was found to inhibit plateletaggregation process in whole human blood.

Methodology: 1) Purification of Lahirinwas achieved byrepeated cation exchange chromatography. 2) Plateletaggregation studies of Lahirin was performed using chro-nolog whole blood aggregometer.

Results: Lahirin was found to inhibit ADP dependentplatelet aggregation process in whole human blood ina dose dependent manner. However, Lahirin did not inhibiteither collagen or ristocetin induced platelet aggregation.Thrombin induced platelet aggregation process was dras-tically slowed down by Lahirin treatment.

Discussions: Lahirin inhibits platelet plug formation byeither binding with P2Y1/ P2y12 receptors or by digestingfibrinogen molecules binding platelets with each other toform platelet plugs. However, the rate of fibrinogen diges-tion by Lahirin is too slow to explain its fast plateletaggregation inhibition activity. As it is specifically inhibit-ing the G protein coupled receptor mediated plateletaggregation process i.e., ADP, Thrombin and Arachidonicacid, Lahirin might be a protease which either directly acton membrane receptors or interferes with the intracellularsignaling molecules of the GPCR signaling pathway.



Fig. 3. Thrombin inducedplatelet aggregationprocess. Blue representsControlHuman whole blood platelet aggregation after thrombin treatment. Blackrepresents thrombin induced aggregation of Lahirin (40 mg) treated blood.

Fig. 4. Time dependent fibrinogenolytic activity of the Lahirin by 10%SDS-PAGE.

Fig. 2. 1) Control Human whole blood 2) 20 mg Lahirin treated blood and3) 40 mg Lahirin treated blood (n¼10).


Keywords: fibrinogenolytic, antiplatelet, peptide10.1016/j.toxicon.2012.04.077

77. Anticoagulant Activity of Moon Jellyfish (Aureliaaurita) Tentacle Extract

Akriti Rastogi, Sumit Biswas, Angshuman Sarkar,Dibakar ChakrabartyBirla Institute of Technology and Science, Pilani - K.K Birla Goa Campus,Department of Biological Sciences, Goa, IndiaE-mail address: [email protected] (A. Rastogi).

Background: Biochemical and pharmacological profileof jellyfish venom is not well studied in jellyfishes foundalong the western coast of India. The venom is expected tocontain novel pharmacologically active molecules of ther-apeutic importance. In this study, Aurelia aurita tentacleextract was investigated for its anticoagulant activity invitro.

Methods: Fibrinogenolytic Assay, Fibrinolytic Assayand Platelet Aggregation Studies using Whole BloodAggregometer.

Results: The Jellyfish Tentacle Extract (JFTE) showedvery strong fibrinogenolytic activity by cleaving Aa and Bbchains of fibrinogen molecule. JFTE completely liquefiedfibrin clots. JFTE appears to contain several anticoagulantproteins or peptides. Some of which appear to be metal-loproteases and some serine proteases. JFTE also inhibitedADP and collagen dependent platelet aggregation, in a dosedependent manner.

Discussion: Purification and characterization of theprotein(s) responsible for anticoagulation is under progress.

Conclusion: JFTE affects the haemostatic system atthree different levels: Platelet aggregation, Fibrinogendigestion and Fibrin clot digestion.

Fig. 1. Dose dependent fibrinogenolytic activity of JFTE. Fibrinogen(2 mg/mL) was incubated independently with different concentrationsof JFTE at 37�C for 180 min. (F) Fibrinogen alone. (JFTE)JFTE alone. Numbers at the bottom of each lane indicates dose of JFTEin mg.


Fig. 2. Inhibition of fibrinogenolytic activity of JFTE by 12% SDS-PAGE. (F)Fibrinogen (2 mg/mL) alonge. JFTE (H) Fibrinogen incubated with 2.5 mg JFTEpre-treated with heat at 100�c for 1 min. (E) Fibrinogen incubated with 2.5mg JFTE pretreated with 2 mM EDTA. (P) Fibrinogen incubated with 2.5 mgJFTE pre-treated with 1 mM PMSF.

Fig. 3. Time depended inhibition of ADP-induced platelet aggregation byJFTE. Positive Control: BloodþADP and Negative Control: Bloodþ0.85% Saline.

Fig. 4. Time dependent inhibition of collagen-induced platelet aggregation by


Keywords: Moon Jellyfish, anticoagulant activity, haemostasis10.1016/j.toxicon.2012.04.078

JFTE. Positive Control: BloodþCollagen and Negative Control: Bloodþ0.85%Saline.

78. Evaluation of the Efficacy of Treatment UsingBothropic or Bothropic/Crotalic Antivenin in Bothropsjararacussu (VIPERIDAE) Experimental Envenomation

Marcio Y. Yano 1, Marcio H. Matsubara 2,Ida S. Sano-Martins 1

1 IBU Instituto Butantan, Laboratório de Fisiopatologia, Brazil2 IBU Instituto Butantan, Laboratório de Farmacologia, São Paulo, BrazilE-mail address: [email protected] (I.S. Sano-Martins).

Background: Snakebites are a public health problem inBrazil. The most effective treatment is antivenin therapyand the effectiveness of Bothrops antivenin for theneutralization of Bothrops jararacussu venom (VBju) has

been discussed by many groups. The aim of this study wasto compare the efficacy of different antivenin treatments inthe experimental envenomation induced by VBju.

Methods: Groups of mice (n¼5) were injected i.v. with 2x Minimum Defibrinogenating Dose (0,50mg/kg) of VBju,or saline (control), and treated with botropic (BA), crotalic(CA) and botropic/crotalic (BA/CA) antivenin, or saline, 1hour (h) after venom inoculation i.v. Blood samples werecollected in 3, 6, and 12 hs after treatment, by the orbitalplexus. Plasma fibrinogen levels (PF) were determined andthe thrombelastography (TE) was carried out on wholeblood samples with the ROTEM� coagulation analyzer. TheNATEM� test was used to assess native whole blood clotformation. Test parameters as clotting time (CT), clottingformation time (CFT), alfa-angle and maximum clottingFirmness (MCF) were acquired for 1 h. Statistical analysiswere accessed by one-way ANOVA and Tukey test.

Results and Discussion: Regarding PF at 3 h, theanimals envenomed and treated with different antiveninshowed a huge variation in the groups. At 12 h, there wereno statistical difference in the PF levels of groups treatedwith saline and that treatedwith antivenins, indicating thatfibrinogen was spontaneously normalized, independentlyof the treatment. On the other hand, at 6 h, the animalstreated with BA/CA showed significantly higher PF levels(1.89� 0.18g/L), when compared with those treated withBA (1.39�0.7g/L), CA (0.93�0.11g/L) or saline (1.17�0.08g/L). PF of all control groups were within normal range (2.48-3.49 g/L). Results of TE are in agreement with the PFobtained from different groups. Thus, at 3 h, most samplesof animals injected with VBju remained unclottable, whileat 12 h, the parameters were significantly restored. More-over, at 6 h, the TE graphic profiles had demonstrated thatanimals treated with BA/CA had a good recovery, showinglower CFT (176.75�28.45 sec.), when compared with thoseof BA (350.66�83.38 sec.), CA (unclottable) and saline(434.25�159.01 sec.).

Conclusion: BA/CA treatment is better than BA in theB. jararacussu experimental envenoming in mice.

Financial support: INCTTOX, CNPq and CAPES.

Keywords: Bothrops jararacussu envenomation, treatment, coagulopathy10.1016/j.toxicon.2012.04.079

79. Hemostatic Disturbances Evoked by Young andAdult Bothrops jararaca Snake Venoms: Analysis of theEnvenoming Process and the Recovery after SpecificAntivenin Treatment

Luana V. Senise 1,2, Sâmella S. Oliveira 2, Marcio Y. Yano 2,Savio S. Sant’Anna 3, Marcelo L. Santoro 2, Ida S. Sano-Martins 2

1Departamento de Fisiologia, Instituto de Biociências, Universidade de SãoPaulo, São Paulo, Brazil2 Laboratório de Fisiopatologia, Instituto Butantan, São Paulo, Brazil3 Laboratorio de Herpetologia, Instituto Butantan, São Paulo, BrazilE-mail address: [email protected] (I.S. Sano-Martins).

Background:Ontogenetic variations in thepro-coagulantenzymes of the young B. jararaca venom (YBjv) makes itmore coagulant than that from adult snakes (ABjv).




Moreover, YBjv is not so efficiently neutralized by specificantivenin in vitro as ABjv. Herein we compared hemostaticdisturbances induced by ABjv and YBjv in rats and assessedtheir recovery after treatment with Bothrops antivenin bythromboelastography (TE).

Methods: Male Wistar rats were inoculated with ABjv,YBjv (1.6 mg/kg, s.c), or saline, and treated with Bothropsantivenin (BA) or saline (100 mL i.v.) 1 hour (h) after venominoculation. Blood samples of not treated rats werecollected 3, 6 and 24 h after injection to evaluate plateletcount (PLT) and plasma fibrinogen (PF). Thromboelastog-raphy (TE) was carried out on citrated (recalcified) andnative (without anticoagulant) blood samples, collected 6and 24 h after injection and treatment with BA or saline.Parameters as clotting time (CT), clot formation time (CFT)and alpha angle were acquired for 1 h.

Results: At 3 h, PLT count of ABjv and YBjv rats dropped,respectively, 13 and 3 times and PF levels fell abruptly (34.0� 2.0 and 42.0 �8.0 mg/dL) in regard to controls (PLT 1058�48.0 x 109/L; PF 252 �8.0 mg/dL,). At 6 h the PF levels stillpretty low. Even so, it could be noticed a slightly recoverytrend, more evident by the PLT count, which increasedsignificantly (ABjv 217.0�89.0 and YBjv 306.0�51.0 x 109/L).At 24 h, the PLT count were much higher (ABjv 624.0 �44.0and YBjv 721.0 �37.0 x 109/L), and the PF consumption levelwas partially reverted (ABjv 94.0 �8.0 and YBjv 57.0 �11.0mg/dL). By TE analysis, at 6 h, it was noticed that the CT, CFT,alpha angle remain greatly altered on non-treated rats,especially in YBjv. At 24 h, these parameters were not totallynormalized, but it was noticed a recovery trend, which isconsistent with the PLT and PF data for this time.

Discussion: With the BA treatment, all the parameterswere significantly restoredespeciallyat24hafter thevenomsinoculation. Thus, despite differences in enzymatic variationsand in vitroneutralizationpotential amongABjv andYBjv, thetreatment with BA was equally efficient in reversing thehemostatic disturbances caused by both venoms.

Financial support: FAPESP, CNPq and CAPES.

Keywords: ontogenetic variation, Bothrops jararaca snake, antivenintreatment, thromboelastography10.1016/j.toxicon.2012.04.080

80. Purification of a Prothrombin Activator fromBothrops moojeni Snake Venom

Marco A. Sartim, Renato C. Caetano, Norival A. Santos-Filho,Wallace de P. Adolpho, Adélia C.O. Cintra, Suely V. SampaioDepartamento de Análises Clínicas, Toxicológicas e Bromatológicas.Faculdade de Ciências Farmacêuticas de Ribeirão Preto- Universidade de SãoPaulo. Ribeirão Preto, SP, BrazilE-mail address: [email protected] (MarcoA. Sartim).

Background:Bothrops snake envenomation is associatedwith several local and systemic effects, inwhich coagulationdisorders are responsible for one of the most severe mani-festations. The involvement of venom proteases capable ofactivate blood coagulation factors and initiate the coagula-tion cascade, leads to blood in coagulability on patients. Theaim of the present work was to isolate a prothrombin acti-vator from Bothrops moojeni snake venom, and assess itsability for thrombin generation and coagulation activity.

Methods: The protease was purified in five chromato-graphic steps, in order, of a size exclusion chromatographyon Sephacryl S-200 column followed by affinity chroma-tography in Blue-Sepharose, hydrophobic interaction inPhenyl-Sepharose, Mono Q ion exchange chromatographyand bioaffinity chromatography using Lentil-LectinSepharose. The non-bonded fraction of the former proce-dure was analyzed in SDS-PAGE and represents the isolatedprotease. In order to evaluate the potential of the toxin onprothrombin activation, amidolytic and proteolytic assaywere performed. The amidolytic assay was carried outincubating human prothrombin and colorimetric substrateS-2238 with several concentrations of the activator.Substrate cleavage by the thrombin generated was moni-tored by measuring the formation of p-nitroaniline. Assaywas also performed after preincubation of the proteasewith15mM of EDTA or benzamidine for 30 minutes at 25oC. Forproteolytic assay, human prothrombin was incubated withthe protease in a molar ratio of 15:1 at 37oC for differenttime periods. The analysis of prothrombin fragments wereperformed in SDS-PAGE under reduction condition. Theinvolvement of B. moojeni activator on coagulation ofnormal and deficient of factor X human plasma wasassessed by recalcification time using turbidimetric assay.

Results: The SDS-PAGE analysis of the purified proteaseshows a protein with w90kDa under non-reduction condi-tions andw70, 18 and 15 kDa domains in the presence of b-Mercaptoethanol. Concerning the protease prothrombinactivation capacity, the amidolytic activity indicates that thetoxin is able to generate active thrombin in a dose-depen-dent manner, being inhibited by EDTA but not benzamidine.Analysis of the proteolytic reaction showed completeconsumption of prothrombin by the protease over the timeinterval, generating thrombin as major fragment. In addi-tion, B. moojeni prothrombin activator was able to inducenormal and factor X deficient human plasma coagulation.

Discussion and Conclusion: The present work describesthe isolation of a possible PIIId classmetalloprotease from thevenom of Bothrops moojeni capable of cleaving prothrombinmolecule, generating thrombin as major product, andinducing human plasma coagulation through coagulationfactor II activation.

Keywords: snake venom, Bothrops moojeni, metaloprotease, prothrombinactivator10.1016/j.toxicon.2012.04.081

81. BaPLA2-IV, a New Type Thrombin Inhibitor,Non Cytotoxic Phospholipase A2 From Bothrops atroxVenom

Adelia C.O. Cintra, P. da Cássio Silva, Marco A. Sartim,Norival Alves Santos Filho, F.Tucci Luiz F., João J. Franco,Suely V. SampaioFaculdade de Ciências Farmacêuticas de Ribeirão Preto - USP, Departamentode Análises Clínicas, Toxicológicas e Bromatológicas, Ribeirão Preto – SãoPaulo, BrazilE-mail address: [email protected] (A.C.O. Cintra).

Background: Snake venoms are composed of biologicalactive molecules that modulates several biological eventssuch as blood coagulation by acting on physiological



Clotting time obtained from the thromboelastometric profile after recal-cification of citrated whole blood samples from chickens preincubatedwith crotalic venom.

ng/mL VP1 VP2 VP3

PBS (Control) (5)100% � 20 %

(6)100% � 20%

(6)100% � 20%

3 (2)110.7 � 27.2

(1)111.2

(1)99.1

30 (5)162.6 � 55.7*p ¼ 0.045

(6)154.3 � 60.6p ¼ 0.063

(6)132.5 � 22.4*p ¼ 0.025

300 (5)409.2 � 208.2*p ¼ 0.011

(5)313.0 � 66.7*p ¼ 0.000

(6)467.2 � 239.6*p ¼ 0.004

330 mL of citrated whole blood samples were preincubated with 20 mL ofcrotalic venom for 5 min at 370C and recalcified with 20 mL of CaCl2 at0.2M. Values are expressed as mean and standard deviation in %,considering the clotting time of samples treated with PBS (control) as100% � 20%; *, p < 0.05 ¼ statistically significant in relation to control(Student`s t test); (), number of experiments.Abbreviations: ng/mL, nanograms per milliliter; VP1, 2 and 3, pools ofcrotalic venoms; PBS, phosphate buffered saline.


components. Thrombin is a key enzyme that participates onblood clotting and cellular carcinogenesis. The present workintents to investigate the involvement of BaPLA2-IV, an acidicphospholipase A2 isolated from Bothrops atrox snake venom,on blood coagulation and cellular proliferation induced bythrombin.

Methods: The PLA2 was isolated from B. atrox venom bythree chromatographic procedure using size exclusion, ionexchange and reverse phase chromatography. The antico-agulant activitywas performed by incubating thrombinwithseveral concentrations of the PLA2 and added of bovinefibrinogen as an initiator of the reaction. The recalcificationtime assay was assessed incubating different concentrationsof PLA2 in humanplasma andmonitored turbidimetrically at650nm. The thrombin clotting time (TCT) was evaluated byincubing human plasma with different concentrations ofBaPLA2-IV with subsequently addition of thrombin, and thetime for clot formation was measured visually. Prothrombintime (PT) and activated partial thromboplastin time (APPT)was performed by assay kits, inwhich several concentrationsof PLA2 was tested. To determine the effect of BaPLA2-IV onplatelet aggregation, the enzyme was pre-incubated withrich platelet plasma (PRP) and added of ADP (5mM) andcollagen (5mg/mL), using aggregometer. In order to evaluateBaPLA2-IV involvement on cell proliferation, severalconcentrations of the toxin was incubated with thrombin,and B16F10 cell line treated with the mixture. The prolifer-ative activity was measured by MTT method.

Results and Discussion: BaPLA2–IV, in low concentra-tions, prolonging the fibrin clotting time. The same activitywas not observed with high concentrations PLA2, in whichreduced coagulation time. A similar effect was observed inrecalcification time assay, showing that BaPLA2-IV acts asan anticoagulant agent in low concentrations. The TCTassay showed that the toxin at 5mg/20mL was capable ofreduce, in w57% clot formation induced by thrombin,prolonging the TCT time. Taking in account PT and APPTassay, at the same concentrations of experiment above,BaPLA2-IV had no apparent effect on both assays. The PLA2was able to inhibit platelet aggregation induced by ADP andcollagen in 82% and 80%, respectively. BaPLA2-IV inconcentrations from 0.01 to 1mg/mL was capable tocompletely inhibit cell proliferation induced by thrombin.

Conclusion: BaPLA2-IV exerts an interesting responsemodulating thrombin activity in different biologicalresponses, as blood clotting and cell proliferation.

Keywords: snake venom, Bothrops atrox, phospholipase A2, thrombininhibitor.10.1016/j.toxicon.2012.04.082

82. Anticoagulant Activity of Crotalic Venoms on WholeBlood Samples from Chickens and Rats

Thayane Ribeiro 1, Benedito C. Prezoto 2, Luciane L. Abbud 2,Nancy Oguiura 1

1 Instituto Butantan, Laboratory of Ecology and Evolution, São Paulo, Brazil2 Instituto Butantan, Laboratory of Pharmacology, São Paulo, BrazilE-mail address: [email protected] (N. Oguiura).

Background: The venom of the rattlesnake Crotalusdurissus has neurotoxic andmyotoxic activities and also can

cause coagulation disturbance. It is constituted mainly bycrotoxin, convulxin, gyroxin and crotamine. This coagula-tion disturbance is mainly due to gyroxin, a serine proteasewith thrombin-like activity. Venom activities can varyaccording to toxin composition or substrate. A Westernblotting analysis showed that crotalic venoms can presentthree different band patterns of gyroxin: two bands of 32.5and 35.5 kDa, one strong or one weak band of 32.5 kDa.

Objective: To analyze if this polymorphism could resultin differences in coagulant activity, we tested the effect ofthe three patterns of crotalic venom on plasma and citratedwhole blood samples from different animal species.

Methods: Three pools of venoms: VP1 (two gyroxinbands), VP2 and VP3 (one strong and one weak band of 32.5kDa respectively) were tested by determining the MinimumCoagulant Dose (MCD) on citrated bovine plasma, as well asby thromboelastometry (TEM) after recalcification of citratedwhole blood samples from chickens and rats.

Results: MCDs on bovine plasma were 92.6, 80.7 and164.7 mg/mL to VP1, VP2 and VP3 venoms, respectively.However, preincubation of all venoms at 300 ng/mL signifi-cantly prolonged the clotting time of recalcified chicken andratwhole blood samples (p< 0.05 using t-test). The observedprolongation of clotting timewas about 3-4 times in chickenwhole blood, whereas in rats was around 1.5 times.

Discussion and Conclusions: The MCDs of crotalicvenoms on bovine plasma observed here are in agreementwith reports published earlier. In addition, the variation ofMCDs suggests an influence of gyroxin polymorphism oncoagulant activity of these venoms, since, VP3 that pre-sented the minor amount of gyroxin showed the highestMCD. Surprisingly, after preincubation of citrated wholeblood samples from chickens and rats with calcium and VP1,VP2 or VP3, all venoms presented anticoagulant activities. Asthese venoms did not inhibit totally the blood coagulation, itindicated that there was no depletion of coagulant factors asfibrinogen or other components as platelets. The anticoag-ulant activities were dose-dependent but the mechanism ofaction is unknown and it was not explored here.

Financial Support: FAPESP, INCTTOX, PIBIC-CNPq.

Table 1


Table 2Clotting time obtained from the thromboelastometric profile after recal-cification of citrated whole blood samples from rats preincubated withcrotalic venom.

ng/mL VP1 VP2 VP3

PBS (Control) (6)100% � 20 %

(6)100% � 20%

(6)100% � 20%

3 (6)116.1 � 37.9p ¼ 0.381

(6)100.4 � 16,7p ¼ 0.974

(5)113,4 � 18,4p ¼ 0.282

30 (6)128.9 � 33.3p ¼ 0.098

(5)111,3 � 21,5p ¼ 0.388

(5)126,5 � 23,2p ¼ 0.072

300 (6)154.8 � 39,4*p ¼ 0,013

(5)131.9 � 13.9*p ¼ 0.009

(5)127,7 � 14,3*p ¼ 0.029

330 mL of citrated whole blood samples were preincubated with 20 mL ofcrotalic venom for 5 min at 370C and recalcified with 20 mL of CaCl2 at0,2M. Values are expressed as mean and standard deviation in %,considering the clotting time of samples treated with PBS (control) as


Keywords: gyroxin, thromboelastometry, minimum coagulant dose10.1016/j.toxicon.2012.04.083

E. History of Toxinology

100% � 20%; *, p < 0,05 ¼ statistically significant in relation to control(Student`s t test); (), number of experiments.Abbreviations: ng/mL, nanograms per milliliter; VP1, 2 and 3, pools ofcrotalic venoms; PBS, phosphate buffered saline.

Fig. 1. Thomas Cream, MD.

83. Clodomiro Picado, Róger Bolaños, and theBeginnings of Toxinological Research in CentralAmerica

José María GutiérrezInstituto Clodomiro Picado, Facultad de Microbiología, Universidad de CostaRica, San José, Costa RicaE-mail address: [email protected].

Review: Snakebite envenomings constitute an importantpublic health issue in Central America. The scientific study ofsnakes, snake venoms, envenomings and their treatment inthis region started with the pioneer work of ClodomiroPicado, an outstanding Costa Rican scientist who worked forthree decades at the Clinical Laboratory of San Juan de DiosHospital, in San José. Picado performed meticulousdescriptions of the Costa Rican snake fauna and character-ized the toxicity of venoms. In addition, he developeda collaborationwith Vital Brazil, of Instituto Butantan, Brazil,which allowed the import of Brazilian antivenoms to CostaRica. Moreover, he promoted a law which enforced landowners to have antivenoms for the treatment of workersbitten by snakes. In 1931, he published Serpientes Venenosasde Costa Rica (Venomous Snakes of Costa Rica), whichsummarized two decades of research. Thirty-five years later,a successful interinstitutional project, known as Programa deSueros Antiofídicos (Antivenom Program), involving theCosta Rican Ministry of Health, the University of Costa Rica,and the Embassy of the United States of America in CostaRica, with the leadership of Róger Bolaños, succeeded inproducing, for the first time in Central America, antivenomsfor the treatment of viperid and elapid snakebite enve-nomings. As a consequence, the Instituto Clodomiro Picadowas founded in 1970, with Bolaños appointed as its firstDirector. Over the years, this institute, which belongs to the

University of Costa Rica, has evolved as an active research,teaching, extension and production center which greatlycontributes to a better understanding of Central Americansnakes and their venoms, to improving the treatment ofenvenomings, to graduate and undergraduate teaching, andto the prevention of snakebites in the region.

Keywords: snakebites, Costa Rica, Clodomiro Picado, Róger Bolaños,antivenoms10.1016/j.toxicon.2012.04.084

84. The Poisoner's Garden: Plants Used to Curse and Kill

Vinodinee L. Dissanayake 1,2

1University of Illinois at Chicago, Department of Emergency Medicine,Chicago, IL USA2 Toxikon Consortium, Cook County Hospital (Stroger), Chicago, IL USAE-mail address: [email protected].

Review: Since the early days of human civilization,poisonings have been a primary mode of murder, andalthough some victims were accidentally poisoned, thosethat are more easily remembered are the intentionalmurders of our wicked past. Although Dr. Hawley Crippenmay have murdered for love, Dr. George Lamson murderedfor money and Dr. Thomas Cream murdered for fun, theyeachused different toxins to do the deed andwere successfuluntil the police of the Scotland Yard caught up to them.Unrelated to these murderers, some victims of plant poisonsinclude Socrates, Georgi Markov, Abraham Lincoln's mother,Nancy Hanks Lincoln, and the armies of Xenophon asreported by Pliny in 401 BCE. In the 1900’s, Henri GirardLandru attempted to murder by mushroom to obtain insur-ancemoney fromhis friends and lovers, while Charles CullenRN murdered patients with the use of many different phar-maceuticals, including digoxin, over the course of fifteenyears before finally being caught. Although plant poisonsnow appear to be an accessory of the past, the long traditionof intentional poisonings have rekindled the intrigue andhorrors of those rueful rendezvous for would-be-assassins ofour modern era. Digitalis was most recently used by LisaLeighAllen tomurder her cophusband in theUS, and Lakhvir“Curry Killer” Singh, a spurned lover in theUK,murdered herex-lover with aconite. Although rarely used today, plantpoisons continue to be some of the most tragic and leastimplicated tools for a murderer or murderess at large.




Fig. 2. Hawley Crippen, MD.

Fig. 4. Nancy Hanks Lincoln.

Fig. 3. Charles Cullen, RN.

Keywords: plants; toxins; poisoners; history10.1016/j.toxicon.2012.04.085

85. TOXIN REVIEWS: The First 30 Years

W. Thomas ShierCollege of Pharmacy, University of Minnesota, Minneapolis, MN, USAE-mail address: [email protected].

Review: During the last 30 years several toxin journalshave come and gone, leaving TOXICON and TOXINREVIEWS as the two enduring scientific journals in thetoxin research field. Organization of TOXIN REVIEWSbegan in 1980 at the invitation of Maurits Dekker,a legendary figure in scientific publishing, co-founder ofInterscience Publishers and father of Marcel Dekker. Thejournal was organized as one of three journals in theJOURNAL OF TOXICOLOGY series with CLINICAL TOXI-COLOGY and CUTANEOUS AND OCULAR TOXICOLOGY. Theseries was intended to build on the strength of CLINICALTOXICOLOGY. Publication by Marcel Dekker, Inc., out ofoffices on two floors of a building in Manhattan, New YorkCity, began in 1982 at two issues/year. In 1989 (Volume 8)Anthony T. Tu, Colorado State University, was added as Co-Editor. In 1991 (Volume 10) the journal increased to threeissues/year, and in 1994 (Volume 14) it increased to fourissues/year. In 2002 (Volume 21) Dr. C. Yu, National TsingHua University, Hsinchu, Taiwan, was added as AssociateEditor. Marcel Dekker, Inc. continued as the publisher ofrecord through 2004 (Volume 23). After Marcel Dekkerretired, his sons ran the company for a few years, then soldit in 2003 to Taylor & Francis, a Division of Taylor & FrancisGroup, an Informa company headquartered in London, UK.Taylor & Francis became the publisher of record in 2005(Volume 24) from offices in Philadelphia, USA. The nameof the journal was changed from JOURNAL OF TOXI-COLOGY - TOXIN REVIEWS to just TOXIN REVIEWS,because their software did not have enough spaces for thewhole name. Name changes are problematic becausea journal's location in libraries (alphabetical order)changes and the journal gets split up in the stacks. In 2007(Volume 26), the production offices were moved back toNew York City and the name of the publisher of record waschanged to Informa healthcare. In 2008 (Volume 27) thecover was redesigned. In 2009 (Volume 28) the AssociateEditor position was replaced with Dr. Hamed Abbas, USDept. of Agriculture and production offices were moved toLondon, UK. The publisher redesigned the cover again andchanged the format to quarto so the journal would fit intotheir Pharmaceutical Sciences series. In 2010 someproduction office activities were moved to Sweden. In2011 Drs. Shier and Tu stepped down, and Dr. R. Man-junatha Kini, National University of Singapore, wasappointed Editor-in-Chief.

Keywords: TOXIN REVIEWS, journal, review10.1016/j.toxicon.2012.04.086


Fig. 2. Caduceus, symbol of the medical profession.


86. From Medicine to Toxinology and Back to Medicine,and the Eternal Role of Snakes in Therapy

Carl-Wilhelm VogelUniversity of Hawaii Cancer Center and Department of Pathology, John A.Burns School of Medicine, University of Hawaii at Manoa, Honolulu, HI, USAE-mail address: [email protected].

Review: The author's work over three decades as anexperimental pathologist involved multiple aspects of thecomplement system, from basic science to its role indisease, to an experimental concept of directing comple-ment activation therapeutically, and to devise a novelconcept of treating diseases with complement pathogen-esis. Much of this work was based on the structural andfunctional homology of Cobra Venom Factor (CVF) tohuman complement component C3, resulting in a human-ized CVF protein as a novel therapeutic agent. At the end, asit turns out, nothing is really new: as multiple scientificmeetings on complement were held in Greece, the authorhad an opportunity to visit the ancient temple in Epidaurus,honoring Asclepius, the demi-god of medicine and healing(Fig. 1). In this healing facility – called the Asclepion, theancient Greek equivalent of a hospital and spa – patientswere treated using snakes, a procedure immortalized in thecaduceus, the symbol of the medical profession to this veryday (Fig. 2).

Fig. 1. Statue of Esclepius, in the museum at Epidaurus, Greece.

Keywords: Asclepius, Caduceus, snakes in therapy10.1016/j.toxicon.2012.04.087

87. Antivenom Production by Instituto Butantan: 110Years of Experience

Jorge KalilButantan Institute, São Paulo, SP, BrazilE-mail address: [email protected].

Instituto Butantanwas founded in 1901 to combat plague.Doctor Vital Brazil was its first director and also a pioneerresearcher on the toxicology of snake venoms. Currently, theInstitute stands out in the public health sector for its scien-tific achievements and the production of vaccines and anti-venom sera, being the largest biopharmacetical producer inLatin America. Its main outputs are vaccines, anti-venoms,anti-toxins, blood products and other biopharmaceuticals.Being a strategic research center, Butantan has partnershipswith several public and private institutions for the devel-opment of new vaccines. The 170 researchers from InstitutoButantan published 325 scientific articles in internationalmagazines with significant impact factors in 2010 alone.Among these studies, it can be highlighted those that screennew bioactive components of poisonous animals. Severalpatents have been filed in this area, indicating the innovativepotential of the institute. Instituto Butantan also stands asscience and technology repository for teachingwith its threescience museums specializing in biodiversity, microbiologyand science history. This is one of the most visited publicparks by foreign tourists in Sao Paulo. The importance andmagnitude of Instituto Butantan as a center for public healthis the result of an intense interaction between research,




production ofmuch needed biopharmaceuticals and culturalactivities,which are critical tomaintaining its excellence. TheInstitute is prime example of the accomplishments of LatinAmerican Science put the service of public health. It hasendured the test of time, survived two dictatorships andcame unscratched 111 years after its foundation as the mostimportant Immunology Center in Brazil.

Keywords: antivenom, Instituto Butantan, immunology, vaccines, bloodproducts, venomous animals10.1016/j.toxicon.2012.04.088

88. Saul Wiener: From Kristallnacht to Toxinology andFragile X.

Kenneth Daniel WinkelAustralian Venom Research Unit, Pharmacology Department, University ofMelbourne, Parkville, Victoria, AustraliaE-mail address: [email protected].

Background: The rise of Nazi Germany led to a tide ofJewish refugees many of who subsequently contributed toscientific advances in the Allied countries. For example,Ernest Chain fled Berlin in 1933 and undertook pioneeringstudies of Australian snake venoms at Oxford beforemoving onto penicillin. Similarly, Wilhelm Feldberg, dis-missed from the Physiological Institute in Berlin in 1933,spent 1936-38 in Australia, examining the tissue responsesto venoms, work that ultimately resulted in the identifi-cation of the leukotrienes. The subject of this presentation,the late Saul Wiener, made major contributions to tox-inology and human genetics. This presentation reviews thelife of this founding member of the International Society ofToxinology, who died in Melbourne on September 15, 2010.

Methods: This biography uses Wieners primary publi-cations, secondary papers about his work, the literature onJewish refugee doctors and scientists in Australia, as well asinterviews with the subject, his wife and his colleagues.

Results: Wiener was born in Germany (July 25, 1923) toparents of Polish origin but migrated to Melbourne afterKristallnacht in 1938 and completed Medicine at theUniversity of Melbourne in 1947. Soon thereafter he enrolledas a PhD student and studied Rheumatic Fever in theDepartment of Microbiology. His 1953 degree, made himequal second, as anAustralianmedical graduate achieving anAustralian PhD. Thereafter, whilst employed as a researchofficer in the Commonwealth Serum Laboratories (1952-58),he developed the Redback spider antivenom and theworld'sfirstmarine antivenom, against Stonefish. He also researchedthe funnel web spider, pioneered the study of Chironexfleckeri box jellyfish venom and first demonstrated thetoxicity of fresh cone snail venom. In the late 1950s he alsoexplored active human immunisationwith snake venom.His1960 MD thesis was probably the first higher degree intoxinology in Australia and included perhaps the firstAustralian toxinology publication in the journalNature. Afterleaving CSL in 1958, Saul's interest in immunology led toa year as a Fulbright Scholar at Columbia University, wherehe developed new skills in chromosome analysis. Returningto Melbourne he commenced as a staff specialist (Allergist)at the Royal Melbourne Hospital. His research moved into

cytogenetics, including some of the earliest work on FamilialX-linked mental retardation (‘Fragile X’).

Conclusion: Like many Jewish German refugees, SaulWiener contributed much to medical science, to his newhome in Australia and thence to the world of broader worldof toxinology.

Keywords: history, toxinology, antivenoms, venom research, Saul Weiner10.1016/j.toxicon.2012.04.089

89. Cobra Venom Factor, an Intriguing VenomComponent: Over 100 Years of Research, and Counting

Carl-Wilhelm VogelUniversity of Hawaii Cancer Center and Department of Pathology, John A.Burns School of Medicine, University of Hawaii at Manoa, Honolulu, HI, USAE-mail address: [email protected].

Review: Cobra Venom Factor (CVF) is the unusual venomcomponent found in the venom of Naja sp. and some otherElapid genera, with most of our knowledge derived from theAsian cobras Naja naja and Naja kaouthia. CVF, strictlyspeaking, is not a toxin. CVF forms a bimolecular enzymewithprey complement component Factor B, called the C3/C5 con-vertase. The CVF-containing enzyme is stable and exhibitsresistance to the complement regulatory proteins, allowing itto continuously activate complement components C3 and C5,leading to the release of the C3a and C5a anaphylatoxins,which are believed to facilitate the entry of the toxic venomcomponents into the bloodstream through an increasedvascular permeability. The anticomplementary activity ofcobra venom was first described in 1903. In 1913, the ana-phylatoxin-generating activity of cobra venomwas described,at a time when the complement origin of the anaphylatoxinswas unknown. After a relative dormancy of 50 years, CVF waspurified as the responsible venom component for bothactivities in the late 1960ies. Subsequently, the molecularinteraction of CVF with the complement system becameunderstood,with CVF simultaneous playing an important rolein unraveling the biochemistry of the alternative complementpathway. Subsequent milestones include the biochemicalcharacterization of CVF (and its convertase (1982)), identifi-cation of its structural similarity to C3 (1984), molecularcloning (1994), recombinant expression (2004), and solutionof its crystal structure, alone and in complex with Factor B(2009). Theobservation that CVF canbe safelyadministered tolaboratoryanimals, causing temporary complement depletion(1970), made CVF awidely used tool over the past 40 years toinvestigate the biological functions of complement in innateand adaptive immunity as well as its role in the pathogenesisof many diseases. Transgenic mice expressing CVF anda permanently low level of complement have been generatedfor the same purpose (2002). CVF was also coupled tomonoclonal antibodies for targeted complement activation,such as killing of tumor cells (1982). More recently, human C3derivatives have been created by replacing short stretches ofsequence with homologous CVF sequence, forming stableconvertases and depleting complement. These human C3derivatives, termed humanized CVF, represent a novel class ofexperimental therapeutics that have been shown to be effec-tive in multiple preclinical models of disease with




complement pathogenesis including paroxysmal nocturnalhemoglobinuria (PNH) (2008), myocardial ischemia reperfu-sion injury (2009), age-related macular degeneration (AMD)(2010), and myasthenia gravis (2011), with no adverse effectsobserved, including in primates (2010).

Keywords: cobra venom factor, complement10.1016/j.toxicon.2012.04.090

F. Insects

90. Proteomic Analysis of the Molecular Targets ofa Peptide from Wasp Venom Through Developing ofan Analytical Platform

Lucilene Delazari dos Santos 1,3, José Roberto Aparecidodos Santos Pinto 2,3, Anally Ribeiro da Silva Menegasso 2,3,Ana Maria Garcia Caviquioli 2,3,Daniel Menezes Saidemberg 2,3, Mario Sergio Palma 2,3

1Center of Studies of Venom and Animal Venomous (CEVAP), University ofSão Paulo State (UNESP), Botucatu, SP; Brazil2 Institute of Biosciences/Department of Biology, Center of the Study of SocialInsects, University of São Paulo State (UNESP), Rio Claro, SP; Brazil3 Instituto Nacional de Ciência e Tecnologia (INCT) em Imunologia / iii, BrazilE-mail address: [email protected] (L. Delazari dos Santos).

Background: The organisms’ answers to changes in theirenvironment or even in their development generate intra-cellular signals which are translated, amplified and con-verted to physiological/ pharmacological answers towardthe signals transduction system from plasmamembrane. G-proteins are responsible for detection of specific andtemporal characteristics of the majority of signal trans-ductionmechanisms. Currently,more than 50% ofmedicinesutilized in the world, act directly or indirectly, by activatingor blocking the G-protein coupled receptors (GPCRs).Therefore, the subject of this study was the development ofan affinity chromatography platform by using the masto-paran peptide Protopolybia-MP III, previously reported asspecific ligand of GPCRs from rat mast cells.

Methods: The peptide Protopolybia MP-III(INWLKLGKAVIDAL-NH2) was synthesized by using F-mocstrategy and then, coupled to the chromatographic resinSepharose 4B, in order to set up an affinity chromatographyprotocol; the column (12 x 2 cm,15mL)was built in TRIS-HClbuffer pH 8.0. Rat peritoneal mast cells were collected andlysedwithNaCl 1M; themembrane extractwas reconstitutedinto proteoliposomes of 300 nm of diameter. The elution ofproteoliposomes suspensionwas carried-out at a flow rate of0.5mL/min andmonitored at 280 nm; fractions of 1mLwerecollected. The liganded proteins (under proteoliposomeform)were removedunder salt gradient from0 to1MNaCl inthe same equilibration buffer. The proteins eluted in eachproteoliposome fraction were extracted with a solution of0.02% (w/v) SDS and submitted to 1-D SDS-PAGE, stainedwith 0.025% (w/v) Coomassie Brilliant Blue and submitted toproteomic analysis. Proteinprofile showedfive protein bandswith molecular weights of 18 to 66 kDa, which were excisedfrom gel, processed and sequenced by ESI-IT-MS/MS.

Results and Discussion: This experimental approach,associated to SDS-PAGE, in gel trypsin digestion and

proteomic analysis, permitted the identification of fiveendosomal proteins: Rho GTPases Cdc 42 and the exocystcomplex component 7- as components of the Caþ2 inde-pendent FcεRI-mediated exocytosis pathway; synapto-somal-associated protein 29 and the GTP-binding proteinRab-3D as components of the Caþ2 dependent FcεRI-medi-ated exocytosis pathway, which are related to cell signaltransduction.

Conclusion: This novel analytical strategy permittedthe identification of some GPCRs, promoting a betterunderstanding of the mast cell activation by peptides fromwasp venom.

Keywords: mastoparan; wasp venom; exocytosis; proteoliposomes; pro-teomics; mass spectrometry; affinity chromatography10.1016/j.toxicon.2012.04.091

91. Proteomic analyses of the Venom from theGiant Ant Dinoponera quadriceps: A Comparative Studyand Characterization of the Major Components of theVenom Derived from 4 Different Areas of Brazil

Camila T. Cologna 1, Jaqueline Cardoso 2,Michel Degueldre 1, Ana P.T. Uetanabaro 3,Eraldo M.C. Neto 3, Edwin de Pauw1, Loic Quinton 1

1 Laboratory of Mass Spectrometry , Université de Liège, Liège, Belgium2 Laboratory of Animal Studies, Universidade do Estado da Bahia, Bahia, Brazil3 Laboratory of Quality Control, Department of Biological Sciences,Universidade Estadual de Feira de Santana, Bahia,BrazilE-mail address: [email protected] (C.T. Cologna).

Background: The order Hymenoptera, which embraceswasps, bees and ants, constitutes the largest group ofvenomous animals. Within the 120,000 already describedspecies, the group of ants represents around 15,000 species.The Ponerinae subfamily is symbolized by theworld’s biggestants (3-4 cm) and is found in subtropical zones of SouthAmerica. In this group of ants, the genus Paraponera e Dino-ponera reveals particularly remarkable venom describingmedical interest. Their sting may produce acute pain, coldsweating, nausea, a vomiting episode, malaise and tachy-cardia. Moreover, peptides isolated from the genus Para-ponera have been described as ion channel modifiers andantimicrobial toxins. Despite those reports, the informationabout the biological properties and composition of theirvenom is still very limited.

Objectives: To study the venom of the giant neotropicalDinoponera quadriceps ant collected in 4 geographicallydifferent regions of Brazil. By using combinatorial massspectrometric approaches, we aim to: (i) characterizing thevenom composition of these ants and (ii) establishinga comparative analysis of the venom from 4 geographicallydifferent regions in Brazil.

Methods: The ants were captured in the surroundingsof Contendas, Manoel Vitorino, Caetite and Feira de San-tana (Bahia, Brazil). Venoms were extracted by mechan-ical stimulation and then dried. An aliquot of each wasanalyzed by MALDI-TOF/TOF (Ultraflex II, Bruker Dal-tonics, Bremen, Germany) and ESI-Q-TOF (Synapt-G1,Waters, Manchester, UK) in direct infusion or witha liquid nanochromatographic separation (nanoACQUITY,Waters, Manchester, UK) step before the MS analysis. In



Table: Systemic effects of centipede envenomations.

Systemic effects Number (%)

Nausea 8 (7.7)


order to determine the cysteine content and the presenceof disulphide bridges, an aliquot of the venom wasreduced (dithiothreitol) and alkylated (iodoacetamide)before being analyzed.

Results: The combinatorial mass spectrometry anal-yses demonstrate that ant venom is a copious source ofnew natural compounds. Several peptides were identifiedand selected for “de novo sequencing”. Regarding to thecomparative study of the 4 regions, we observed slightdifferences in the expression of some peptides and eventhe absence of others. The analysis of the reduced andalkylated venom carried out until now revealed thatthe venom, as expected, did not hold peptides withcystines.

Conclusion: This study has reported for the first timethe mass fingerprint of Dinoponera quadriceps venomderived from 4 areas of Brazil. Further characterization ofthe compounds is being made for a better understanding ofits biological properties.

Keywords: proteomics, ant venom, MS10.1016/j.toxicon.2012.04.092

Vomiting 6 (5.8)Rash 4 (3.8)Fever 2 (1.9)Systemic swelling 2 (1.9)Palpitations 2 (1.9)Abdominal pain 1 (1)Wheezing 1 (1)Chest tightness 1 (1)Flushing 1 (1)Itchiness without rash 1 (1)

92. Centipede Envenomation: 104 Cases from Bangkok,Thailand

Rittirak Othong 1,2, Winai Wananukul 3, Rais Vohra 4,5

1Department of Emergency Medicine, Bangkok Metropolitan AdministrationMedical College and Vajira Hospital, Bangkok, Thailand2Centers for Disease Control, Atlanta, GA USA3Department of Medicine, Ramathibodi Hospital, Mahidol University,Bangkok, Thailand4Department of Emergency Medicine, UCSF Medical Center, Fresno, CA USA5California Poison Control System, Fresno-Madera Division, Fresno, CA USAE-mail address: [email protected] (R. Vohra).

Introduction: This study describes epidemiology, clin-ical manifestations, and various treatments for enveno-mation by centipedes in patients presenting to an urbantropical emergency department in Bangkok, Thailand.

Methods:We retrospectively analyzed cases of centipedestings admitted to BMAMC/ Vajira Hospital, Bangkok,between 2004 and 2009; data were collected on demo-graphics, local and systemic effects, treatments, complica-tions, and disposition.

Results: There were 104 cases included in this study. 52%of patients were female. Mean age was 28 years (range, 1month to 76 years). Most of the patients (77%) had nounderlying diseases. Median time to ED presentationwas 40minutes (range, 15 minutes to 48 hours). Most injuries(85.9%) occurred between 6 pm and 6 am. Injury incidencewas highest in April and May, and again in October throughDecember. Envenomation sites were recorded in 96% (100/104) of patients; nine patients were stung more than once.Feet (29.4%) and hands (22.9%) were the most commonlyinjured sites. Local effects were common: 96% of patientsreported localized pain, and 78% had localized swelling.Systemic effectswere found in 12 cases (11%); symptoms andsigns included nausea, vomiting, rashes, fever, systemicswelling, palpitations, abdominal pain, wheezing, chesttightness, flushing, and pruritus (see table). Anaphylaxis wasdiagnosed in 3 cases.

Patients were treated primarily for pain control, with98.1% receiving analgesic drugs, and 33.7% treated with localanesthesia. Prophylactic antibiotics, tetanus immunization,antihistamines, and steroids were prescribed in 73%, 28%,24.%, and 10% of cases, respectively. Return ED visits wererecorded for 5 patients (4.8%), for worsening local effects,gastroenteritis, or vertigo. Only one case developed cellulitisand received intravenous antibiotics. There were no deaths.

Conclusions: Nearly all patients had local effects, whilesystemic effects were rarely observed and resolved withminor supportive care. The occurrence of hypersensitivityreactions in 3 patients suggests that, in a minority ofpatients, centipede venom may, like hymenoptera venom,be immunogenic. More than half of patients presented inthe evening to an urban hospital within 1 hour of enve-nomation, confirming the synanthropism and nocturnalbehavior of centipede species in urban Thailand.

Keywords: centipede envenomation; Thailand; venomous arthropods10.1016/j.toxicon.2012.04.093

93. Mechanisms Implicated In Cell Proliferation and CellSurvival Induced By Recombinant Losac, A Cell AdhesionMolecule From Lonomia oblique

Miryam P. Alvarez-Flores 1,2, Cesar M. Remuzgo 3,Yara Cury 2,3, Rosemary V. Bosch 1, Beatriz B. Vaz-De-Lima 1,Durvanei A. Maria 1, Ana M. Chudzinski-Tavassi 1,21Biochemistry and Byophysics Laboratory, Butantan Institute, Sao Paulo, Brazil2CAT-CEPID, Butantan Institute, Sao Paulo, Brazil3 Special Laboratoryof PainandCell Signaling,Butantan Institute, SaoPaulo, BrazilE-mail address: [email protected] (M.P. Alvarez-Flores).

Background: Losac is the first members of the Ig-likeimmunoglobulin superfamily exhibiting proteolytic func-tion. It was purified from the bristle extract of the caterpillarof Lonomia obliqua (Lepidoptera). Previously, we have shownthat purified Losac is capable to induce cell proliferation, cellsurvival and the liberation of hemostatic mediators inendothelial cells. However little is known about the mecha-nism and structural features implicated in these activities. Asan initial step towards the functional characterization ofLosac,we found the clonewith the transcript coding for Losacthrough the screening of a bristle's cDNA library.

Methods: Recombinant Losac (rLosac) was expressed inE. coli BL21(DE3). Several cell cultures and methodologieswere applied aiming to unravel molecular mechanismsmodulated by rLosac. Cell viability and mitochondrial




metabolic functions were assessed using MTT assay. Cellcycle was analyzed by flow cytometry. Nitric oxide (NO)liberation was determined in cell supernatants according tothe Griess reaction. The content of tissue plasminogen acti-vator (t-PA) in the supernatant was measured with an ELISAkit. Cell signaling studies were analyzed byWestern blotting.

Results: In HUVECs (human umbilical vein endothelialcells), rLosac elicits the same activities observed for thenative Losac purified from bristles: cell proliferation, cellsurvival and liberation of NO and t-PA. Moreover, in a quies-cent state, rLosac-induced cell proliferationwas inhibited bystaurosporine (a PKC inhibitor) but no by L-NAME (aninhibitor of eNOS) or LY294002 (a PI3K inhibitor). Westernblotting analysis of quiescent cells showed the presence ofphospho-p44/42MAPKs in the basal state and an increase intheir levels by rLosac treatment. In DRG (dorsal root ganglia)neurons, no morphological effects (in number and appear-ance) were observed after treatment with rLosac (10-200nM). Instead, rLosac was able to significantly increase themitochondrial function. This activity was inhibited byLY294002 and PD98059, both inhibitors of the PI3K andMAPK signaling pathways respectively. In fibroblast (FN-1),rLosac protected cells from serum-deprived apoptosis.

Discussion: All these results indicate that rLosac triggercell viability through the activation of survival pathways inthe early hours of cell contact with Losac and subsequentgene expression, DNA synthesis and proliferation.

Conclusion: A potential biotechnological application ofrLosac is minimizing cell death of animal cell culture(production purposes) and consequently increasing theproductivity.

Financial Support: FAPESP, CNPq, CAT-CEPID.

Keywords: Lonomia, cell adhesion molecule, cell proliferation, cell survival10.1016/j.toxicon.2012.04.094

94. Bee Venom and Pain

Jun Chen 1,2

1 Institute for Biomedical Sciences of Pain, Tangdu Hospital, The FourthMilitary Medical University, Xi'an, China2 Institute for Biomedical Sciences of Pain, Capital Medical University, Beijing,ChinaE-mail address: [email protected].

Review: In this presentation, I will summarize a 15-yearhistory of the studies on the nociceptive effects of bee venomand its main active polypeptide melittin inmammals. In thisseries of studies, behavioral assays, electrophysiologicalrecordings, pharmacological and neurochemical approacheswere used. It has been revealed that melittin, as a uniquealgogen, on one hand, acts on phospholipase A2 (PLA2)–lipoxygenases (LOXs) / cyclooxygenases (COXs) pathways,resulting in activation and sensitization of transient receptorpotential vanilloid receptor 1 (TRPV1) by LOXs/COXs-medi-ated products in primary nociceptive sensory neurons. Onthe other hand, melittin can activate TRP canonical (TRPC)channels probably through diacylglycerol (DAG), potentTRPC3/6/7 channels activator, produced by G-protein coupledreceptors (GPCRs)-mediated phosphorylation of PLC thatmight involve ATP P2Y receptors in the periphery. Activation

of TRPV1 and TRPCs leads to tonic firing of primary noci-ceptive sensory neurons that results in long-term plasticchanges (central sensitization) in the spinal dorsal hornwhere plays very important roles in driving persistentspontaneous nociceptive behaviors and pain hypersensi-tivity induced by subcutaneous injection of bee venom andmelittin (mimicking bee sting injury).

Keywords: bee venom, melittin, pain10.1016/j.toxicon.2012.04.095

95. Biological and Immunochemical Characterization ofPremolis semirufa Caterpillar-Bristles Toxic Components

Isadora M. Villas-Boas 1, Rute M. Gonçalves-de-Andrade 1,Giselle Pidde-Queiroz 1, Suely L.M.R. Assaf 2,Fernanda C.V. Portaro 1, Osvaldo A. Sant'Anna 1,Carmen W. van den Berg 3,1, Denise V. Tambourgi 11 Immunochemistry Laboratory, Butantan Institute, São Paulo, SP, Brazil2Genetics Laboratory, Butantan Institute, São Paulo, SP, Brazil3Department of Pharmacology, Oncology and Radiology, School of Medicine,Cardiff University, Cardiff, UKE-mail address: [email protected] (D.V. Tambourgi).

Background: Pararama, the popular name of the larvalform of the moth Premolis semirufa inhabits rubber planta-tions in the Amazon region and the accidental contact of theskin with the caterpillar's bristles or cocoons results inimmediate and intense heat, pain, edema, and itching. Inmany cases a chronic inflammatory reaction with immobi-lization of the joints occurs. Despite being a serious problemin occupational medicine and a social problem affecting theBrazilian Amazon region, since the rubber tappers can nolonger return to their activities, which are the source of theirlivelihood, studies on the pathogenesis of Pararama arescarce. Thus, the aim of the present study was to analyze thebiological and immunochemical characteristics of Premolissemirufa caterpillar's bristles crude extract.

Results: Analysis of the bristles extract in in vitro assaysrevealed the presence of proteolytic and hyaluronidaseactivities but no phospholipase A2 activity. In vivo assays,using mice, showed that the extract was not lethal, butcaused significant edema and induced intense infiltrationof inflammatory cells to the envenomation site. Further-more, the bristles components stimulated an intense andspecific antibody response but autoantibodies such as anti-DNA or anti-collagen type II were not detected.

Conclusions: Together, these data show the existence,in the Premolis semirufa’s bristles extract, of a mixture ofdifferent enzymes that may be acting together in thegeneration and development of clinical disease manifes-tations. Moreover, this study demonstrates the productionof high antibody titers in mice inoculated with the extract,which may also contribute to the genesis of the inflam-matory reactions observed in the envenomation. Theabsence of autoantibodies indicate that the molecularmechanisms causing disease after multiple contact withthe Premolis semirufa’s bristles differ from that observed inchronic synovitis, such as the rheumatoid arthritis. Thebristles toxic action, high antibody response with theformation of immune complexes and complement




activation may also play a role in the establishment of thedisease. These aspects will be further investigated in futurestudies.

Keywords: Premolis semirufa, Pararama, caterpillar, inflammation10.1016/j.toxicon.2012.04.096

96. Chemical and Biological Characterization of a NovelNeuropeptide in the Venom of Solitary Digger Wasp

Ken-ichi Nihei 1, Kohei Kazuma 2, Kenji Ando 2,Katsuhiro Konno 2

1 Faculty of Agriculture, Utsunomiya University, Utsunomiya, Japan2 Institute of Natural Medicine, University of Toyama, Toyama, JapanE-mail address: [email protected] (K. Konno).

Background: Solitary wasps are known to inject theirvenoms into insects or spiders, paralyzing the prey in orderto feed their larvae. Therefore, the solitary wasp venomsshould contain a variety of neurotoxins acting on nervoussystems. In fact, polyamine toxins, peptide neurotoxins anda protein paralyzing toxin have so far been found in severalsolitary wasp venoms. Besides the neurotoxins, we havefound that cytolytic peptides are also present in the solitarywasp venoms. In our continuing survey of biologicallyactive substances in solitary wasp venoms, we foundFMRFamide-related peptides in the venoms of the solitarydigger wasps Sphex argentatus argentatus and Isodontiaharmandi. Reported herein are the isolation, sequencingand biological evaluation of these neuropeptides.

Methods: The venom extracts with 50%MeCN/H2O/0.1%TFA were purified by reverse-phase HPLC, and theisolated peptides were chemically characterized by massspectroscopy.

Results: Analysis of MS/MS spectra by MALDI-TOF/TOFrevealed the sequence of a novel peptide as EDVDHVFLRF-NH2, which is highly homologous to leucomyosupressin(pQDVDHVFLRF-NH2) and SchistoFLRFamide (PDVDHVFLRF-NH2), the FMRFamide-relatedneuropeptides from cockroachand locust, respectively. Indeed, this new peptide inhibitedcontraction of the locust oviduct at 50 nM with the sameextent as SchistoFLRFamide. A non-amidated peptide（EDVDHVFLRF）was also isolated, but this showed noactivity in the locust oviduct contraction.

Conclusions: This is the first example of FMRF-relatedneuropeptide to be found in solitary wasp venom.

Keywords: solitary wasp venom, neuropeptide, FMRFamide-related peptide10.1016/j.toxicon.2012.04.097

97. Insulin-Binding Protein-Like Venom Protein of theSolitary Hunting Wasp, Eumenes pomiformis(Hymenoptera: Eumenidae)

Ji Hyeong Baek, Si Hyeock LeeDepartment of Agricultural Biotechnology, Seoul National University, Seoul,Republic of KoreaE-mail address: [email protected] (J.H. Baek).

Background: Insulin/insulin-like peptide-binding protein(IBP) and its transcript are abundantly found in Eumenes

pomiformis venom and the venom gland/sac-specific ESTlibrary, respectively.E.pomiformis IBP (EpIBP) ismost similar toinsect IBP-likeproteins thatareknownto inhibit insect growthand insulin signaling. To investigate the toxicity of EpIBP, invivo expressed EpIBP was injected into lepidopteran larvae.

Methods: EpIBP was expressed by Escherichia coli. EpIBPin the inclusion body was unfolded and then refolded. Thethird instars of Spodoptera exigua were fixed in an incisedsilicone tubing, and the expressed EpIBP was injected intothe abdominal cavity (0.5 mg/0.5 ml/larva) using a nano-injector and a glass capillary.

Results and Discussion: S. exigua larvae that wereinjected with the in vivo expressed EpIBP showed a 20%lower pupation rate than the control larvae at the fifth dayafter the injection, although their body weight was notsignificantly different to the control when the larvae wereprovided artificial diet after the injection. EpIBP extendedthe larval stagewithout inducing paralysis of S. exigua larvae.To investigate the effects of EpIBP on caterpillar undera starvation condition, survivorship and body weight of theEpIBP-injected larvae that were not provided artificial dietafter the injection were observed until all the tested larvaedied. The survivorship of the EpIBP-injected larvae was 24%and 36% higher than the control larvae at the fourth and fifthday after the injection, respectively. The body weight of theinjected larvae also showed a statistically significant differ-ence under the starvation condition compared with thecontrol. At the fourth day after the injection, the bodyweightof the control larvae reduced to 59%, which is approximately10% lower than that of the EpIBP-injected larvae. It has beenreported that IBP ofDrosophilamelanogaster (Imp-L2) and itshomolog Sf-IBP from S. frugiperda repress the insulin/insulin-like peptide signaling pathway. These results suggestthat EpIBP might inhibit the metabolism of the caterpillars,which is likely relatedwith the insulin-like peptide signalingpathway, suppress the loss of body weight and eventuallyextend the larval stage.

Conclusion: This finding suggests that the large amountof IBP in E. pomiformis venom likely functions to suppressthe larval growth of prey and to allow the prey to endurestarvation under paralysis conditions.

Keywords: venom, insulin-binding protein, growth regulation10.1016/j.toxicon.2012.04.098

G. Marine

98. Transcriptomic and Peptidomic Characterisation ofthe Conus marmoreus Venom: Insights on ConopeptideDiversity and Venomic Processing

Ai-hua Jin, Sébastien Dutertre, Quentin Kaas,Richard J. Lewis, Paul F. AlewoodThe Institute for Molecular Bioscience, The University of Queensland, St Lucia,Queensland, AustraliaE-mail address: [email protected] (A.-h. Jin).

Background: Next generation sequencing technologiesare revolutionising venom peptide discovery with anunprecedented speed and coverage. As of today, 3




Fig. 1. Relative intensity (%) v. Time (min).


transcriptomes of cone snail venom ducts have been pub-lished, and surprisingly only a limited number of genesencoding these bioactive peptides (< 100 per species) wererecovered for each species. Therefore, there is a strikingdisparity between the number of transcriptomic sequencesand the number of peptides (w 200 to> 1,000) reported pervenom fromMS studies. In the present study, we have inte-grated for the first time next generation sequencing withproteomic technologies to comprehensively define the con-opeptide composition of the venom from a single species ofConidae (Conus marmoreus).

Methods: A total of 92 conopeptide precursor sequencesfrom 16 gene families (Superfamilies) were extracted fromtranscriptomic data using sequence similarity search toolsand phylogenetic analyses.

Results: Using the highly sensitive 5600 TF MS instru-ment, we identified > 3000 unique peptides from onesingle LC-ESI-MS/MS run. All conopeptides derived fromtranscriptomic sequences could be matched to mass spec-trometry data at the MS level within 100 ppm accuracy,from which 69 (75%) had series of partial or complete MS/MS coverage confirming these matches.

Conclusions: This study reveals that conopeptidediversity arises from a more limited set of genes, mainlythrough alternative cleavage sites, residue-specific post-translational modifications, and selective N- and C-terminal truncations. This integrated strategy is expectedto accelerate the discovery of drug leads from cone snailvenoms.

Keywords: conopeptide, transcriptomic, proteomic10.1016/j.toxicon.2012.04.099

99. The Venom of Conus geographus Revisited

Sébastien Dutertre, Ai-hua Jin, Paul F. Alewood,Richard J. LewisDivision of Chemistry and Structural Biology, Institute for MolecularBioscience, The University of Queensland, Brisbane, AustraliaE-mail address: [email protected] (S. Dutertre).

Background: Conus geographus deservedly has a repu-tation as the most dangerous of all cone snails, withreported human fatality rate as high as 65 % . While crudevenom gland extracts have been used for the past 50 yearsto determine animal LD50 and isolate some potent paralytictoxins, the venom actually injected by C. geographus into itsvictim has never been studied. We hypothesised that theinjected venommay prove different from the crude venomextracted from the venom duct, as recently demonstratedfor other cone snail species (1-3).

Methods: To test this hypothesis we developed for thefirst time a milking procedure that allows venom collectionfrom this species. Venom samples were then analysedusing LC-MS methods.

Results: There was a linear correlation between thevolume and dryweight of venom injected and the size of theshell. We also report on the molecular composition andintraspecific variations of the injected venom, and demon-strate significant differences between injected and dissected

venoms from the same individual. Finally, extrapolation ofour data to an historic fatal case of C. geographus enveno-mation allowed the determination of the human lethal dose,which may help in the management of future victims.

Conclusions: The injected venomof C. geographusprovedto be a highly complex mixture of mainly small molecularweight compounds. Significant intraspecific variations werenoted, as well as major differences between milked anddissected venoms. Since it is directly relevant to humanenvenomations, further research on the injected venom isneeded to uncover the associated lethal biological effects.

References

Biass, D., Dutertre, S., Gerbault, A., Menou, J. L., Offord, R.,Favreau, P., and Stocklin, R. (2009) Comparative proteomicstudy of the venom of the piscivorous cone snail Conusconsors. J Proteomics 72, 210-218.Dutertre, S., Biass, D., Stocklin, R., and Favreau, P. (2009)Dramatic intraspecimen variations within the injectedvenom of Conus consors: an unsuspected contribution tovenom diversity. Toxicon 55, 1453-1462.Jakubowski, J. A., Kelley, W. P., Sweedler, J. V., Gilly, W. F.,and Schulz, J. R. (2005) Intraspecific variation of venominjected by fish-hunting Conus snails. J Exp Biol 208, 2873-2883.

Keywords: Conus geographus, proteomics, injected venom10.1016/j.toxicon.2012.04.100

100. The Nematocyst Venom of Hydra: What is itComposed of and How Did it Evolve?

Tamar Rachamim, Eliezra Glasser, Daniel SherDepartment of Marine Biology, Leon H. Charney School of Marine Sciences,University of Haifa, IsraelE-mail address: [email protected] (D. Sher).

Background: The genomes of cnidarians encode manyputative toxins.However, to date, it isnot clearwhichof thesetoxins are injected through the nematocysts and which arefound in other tissues. Here, we characterize the nematocystvenom of the model cnidarian Hydra magnipapillata andtrace the genomic underpinnings of the venoms evolution.

Methods: We identified soluble components from iso-lated nematocysts of H. magnipapillata using shotguntandem mass spectrometry. Bioinformatic analyses wereperformed to compare the identified venom components




with the arsenal encoded in the Hydra genome, and thebiological activity of selected proteins was verified byrecombinant expression.

Results and Discussion: We identified 162 proteins inthe soluble fraction of Hydra nematocysts, of which 25encode putative toxins. These include potential neurotoxinsaffecting Kþ channels, phospholipase A2s (PLA2s) andseveral groups of hemolysins, but no orthologs of the well-studied anemone peptide neurotoxins affecting Naþ chan-nels. Venom proteins seem to have evolved through severaldistinct mechanisms: the actinoporin gene family diversi-fied strongly in Hydra, with at least two of six membersmaintaining hemolytic activity despite significant changesin sequence compared to similar toxins from sea anemones(e.g. equinatoxin 2). In contrast, type III PLA2s represent anancient gene family of which several members wererecruited to the venom, with the venom isoforms fromdistantly related organisms revealing signs of convergentevolution likely representing similar selective pressure.Finally, several venom proteins contain an ShK domainsimilar to Kþ channel toxins, as well as other domainsassociated with venom in multiple organisms such as CRISP,C-type lectin and Zn-metalloprotease. In all cases, the ShKdomains are encoded by a separate exon, suggestinga mechanism whereby exon shuffling provides a “venomrecruitment shortcut”: secreted proteins into which theseexons are integrated will potentially bind Kþ channels foundon excitable tissue – a major target for neurotoxins. Ourresults paint a dynamic picture of the evolution of cnidarianvenom and highlight a potential mechanism for generatingmulti-functional venom proteins from the available pool ofmolecular “building blocks”.

Keywords: Hydra, nematocysts, mass spectrometry, exon shuffling, geneduplication, convergent evolution10.1016/j.toxicon.2012.04.101

101. Manipulation of Zoanthids in Home MarineAquarium: Unexpected Cause of Intoxication Involvinga Whole Family

Marieke A. Dijkman, Irma de VriesNational Poisons Information Center, University Medical Center, Utrecht, TheNetherlandsE-mail address: [email protected] (M.A. Dijkman).

Background: A new danger has recently been identifiedin marine aquaria as highly toxic Palythoa zoanthids haveentered the home marine aquaria trade. The Dutch PoisonsInformation Center was recently involved in a palytoxinpoisoning of all members of one family.

Case report: Two adults and 2 teenagers were pre-sented at the emergency department with nausea, head-ache, fits of dry cough, shortness of breath, chest pain,tachycardia and fever. Approximately three hours earlier,the father attempted to destroy a zoanthid colony (about 10polyps) using boiling water. Immediately a foul odour wasnoticed and within one hour, all members of the familyexperienced signs of intoxication. All victims were hospi-talised. On examination, a decreased PO2 saturation wasnoticed and elevated leukocyte count was found. Repeated

chest radiogram showed no infiltrates. Treatment consistedof O2 administration, nebulised salbutamol and acetamin-ophen. During the following days, symptoms graduallysubsided and all patients were discharged 2 days later.

Results:Analyses of theDutchPoisons InformationCenterdatabase revealed 7 previous cases related to presumedpalytoxin exposure after handling/elimination zoanthids inhome marine aquaria. The first consultation was in 2006. Infive cases symptoms developed after dermal/parenteralcontact and in 2 cases after inhalation of aerosols. Symptomsvaried from dizziness, nausea, headache, shivering, cold-warm sensations, malaise, paresthesia, numbness especiallyof the hands/arms, tomild tomoderate dyspnoe andmyalgia.Contact dermatitis was seen after dermal contact.

Discussion: At present, intoxications by palytoxinare identified on the basis of symptoms. Identification ofpalytoxin in serum/urine of the patient or in zoanthids isvery difficult. The major concern is finding a lab capable ofanalysing the samples.

Conclusions: As highly toxic Palythoa zoanthids haveentered the home marine aquaria trade in Europe includingthe Netherlands, Poisons Information Centers need to beawareof the circumstancesunderwhichpalytoxincan induceintoxications. Efforts shouldbemade to be able to identify thesource of intoxication, using human and zoanthids samples.

Keywords: zoanthid, intoxication, aquaria10.1016/j.toxicon.2012.04.102

102. Convergent Evolution of Sodium ion Selectivity inMetazoan Neuronal Signaling

Maya Gur Barzilai 1, Adam M. Reitzel 2,Johanna E.M. Kraus 3, Dalia Gordon 1, Ulrich Technau 3,Michael Gurevitz 1, Yehu Moran 3

1Department of Plant Molecular Biology and Ecology, George S. Wise Facultyof Life Sciences, Tel-Aviv University, Tel-Aviv, Israel2Biology Department, Woods Hole Oceanographic Institution, Woods Hole,MA, USA3Department for Molecular Evolution and Development, Faculty of LifeSciences, University of Vienna, Vienna, AustriaE-mail address: [email protected] (M.G. Barzilai).

Review: The ion selectivity of metazoan voltage-gatedNaþ-channels is critical for neuronal signaling and has longbeen attributed to a ring of four conserved amino acids(DEKA) that constitute the ion selectivity filter at thechannel pore. The sea anemone Nematostella vectensis isa member of the early-branching venomous metazoanphylum Cnidaria and has five genes encoding Naþ-channelhomologs. Expression and characterization of these chan-nels in Xenopus oocytes revealed that four of them prefer-ably conduct calcium ions, whereas one channel isNaþ-selective despite a non-consensual DKEA selectivityfilter. Mutagenesis and physiological assays indicated thatpore elements additional to the selectivity filter determinein this channel the preference for Naþ ions. Phylogeneticanalysis shows that Naþ-channel homologs have alreadybeen present in unicellular organisms and are found inmost animals, but vertebrates. Naþ-selective channelsbearing the DEKA selectivity filter appeared first in the




urbilaterian and exists in most 'higher' animals (e.g.,mollusks, insects, chordates). The Naþ-selective channel ofNematostella is clustered within a channel group unique toCnidaria, which diverged >500 million years ago fromCa2þ-conducting Naþ-channel homologs. The identificationof cnidarian Naþ-selective ion channels distinct fromthe channels of 'higher' animals (Bilateria) indicates thatselectivity for Naþ in neuronal signaling emerged inde-pendently in these two animal lineages, and might haveanswered needs of higher complexity and faster, moreaccurate neuronal signaling that would be separated fromintracellular signaling mediated by Ca2þ ions. These find-ings change the common view about the evolution andmolecular requirements for Naþ ion selectivity.

Keywords: Naþ-channels, ion selectivity, evolution10.1016/j.toxicon.2012.04.103

103. Emergent Marine Toxins in Europe: New Challengesfor Scientists and Regulatory Authorities

Vitor Vasconcelos 1,2, Mafalda Batista 2, Rosa Cianca 2,3,Joana Azevedo 2,4, Marisa Silva 1,2

1Department of Biology, Faculty of Sciences, Porto University, Portugal2Marine and Environmental Research Center – CIIMAR/CIMAR, PortoUniversity, Portugal3 Faculty of Biology, University of Vigo, Spain4Health Technology School, Porto Polytechnic Institute, Porto, PortugalE-mail address: [email protected] (V. Vasconcelos).

Review: The most common HAB (Harmful Algae Bloom)toxins are regulated and monitored in European countries,which has decreased the risk of shellfish consumptionregarding PSP (Paralytic Shellfish Poisoning), ASP (AmnesicShellfish Poisoning) and DSP (Diarrhoeic ShellfishPoisoning). The monitoring of these toxins in bivalves isestablished since many decades ago, but new toxin vectorsare being found, including gastropods and equinoderms,that are edible but not regulated. Other emergent toxinssuch as cyclic imines (spirolides, gymnodimines, pinna-toxins and pteriatoxins) are being studied but their toxicityand prevalence is not known and there are no regulatorylimits in Europe or anywhere in the world. There are alsoincreasing reports on other emergent marine toxins oftropical origin such as tetrodotoxin (TTX), ciguatera (CTX)and ovatoxin and palitoxin analogues (PTX) in Europeanwaters. Most of these are very toxic by oral route attaininghigh levels in vertebrates or invertebrates that enter humanfood chain. The existence of migratory routes for marinespecies via Suez Canal and the global warming effects maybe some of the causes accelerating these processes.Recently, the production of the neurotoxic amino acid b-N-methylamino-L-alanine (BMAA) by marine and estuarinecyanobacteria isolated from Portuguese coastal environ-ments has called our attention due to the potential impli-cation on human neurodegenerative diseases. There isevidence that BMAAmay also be taken up by food chains inthe Baltic Sea, with the highest levels in the brain andmuscle of bottom dwelling fishes. We detected BMAA inseveral strains of cyanobacteria isolated from estuarineenvironments in levels up to 63 mg/g dry weight. BMAA

should be further investigated namely inwhat concerns themain vectors and toxin levels to perform an integrative riskanalysis. In this work, we focus on the emergent marinetoxins detected in Europeanwaters giving special attentionto those that have serious implications in human health.The most recent chemical and biological analyticalmethods available enable us to detect lower amounts oftoxins in more complicated matrices. Chronic toxicitydata is needed in order to allow the establishment ofTolerable daily intake (TDI) for some of these “new” toxins.The seasonal and geographical occurrence of these toxinsshould be cleared so as to allow us to implement accuraterisk analysis and help regulatory authorities to establishnew guideline values.

Financial support: This work was supported by theInterreg Projects ATLANTOX and PHARMATLANTIC and bythe FCT project PTDC/MAR/099642/2008.

Keywords: emergent toxins, marine environment, Europe10.1016/j.toxicon.2012.04.104

104. New Actinoporin from the Northern Pacific SeaAnemone Urticina crassicornis

Andrej Razpotnik 1, Peter Ma�cek 1, Igor Kri�zaj 2, Tom Turk 1

1Department of Biology, Biotechnical Faculty, University of Ljubljana,Ljubljana, Slovenia2Department of Molecular and Biomedical Sciences, Josef Stefan Institute,Ljubljana, SloveniaE-mail address: [email protected] (T. Turk).

Background: Actinoporins are well known 20 Kda basiccytolytic proteins that had been isolated from about 30species of sea anemones. We report on isolation, primarysequence and initial characterization of novel actinoporinfamily member from Urticina crassicornis.

Methods: living sea anemones were milked and theirexudate concentrated. Afterwards, proteins in the exudatewere separated by the means of gel and ion-exchangechromatography. Fractions were analyzed by SDS-PAGEand had their hemolytic activity determined via a turbidi-metric method. The fraction containing pure cytolysinnamed UcT 1 was subjected to N-terminal sequencing.RACE-ready cDNA was prepared from the isolated mRNAusing the GeneRacer Kit (Invitrogen), after which 5’ and 3’rapid amplifications of cDNA ends (RACE) were performed.Primer and the degenerate primer corresponding to theamino acid sequence obtained by Edman degradation ofthe wild-type UcT were used. The constructs were ampli-fied by PCR. Products of the PCR reactionwere separated byMini agarose electrophoresis and the band of interest wasextracted from the gel and cloned with the GeneJET PCRCloning Kit (Fermentas), using E. coli DH5a chemicallycompetent cells. Positive clones were determined byrestriction analysis and sequenced. Primers used for 5’RACEwere the gene specific reverse primer TGCACGGCTGA-CACGAATTTGC and the GeneRacer 5’ Primer, both in 1 mMfinal concentrations. PCR was performed with the samereaction mix as for the 3’RACE reaction. All obtainedsequences were processed, analyzed and aligned with theVector NTI software package (Invitrogen). The isoelectric




point for the obtained protein was determined and itsinsertion into lipid monolayers was studied.

Results: UcT I shows a typical actinoporin sequencelargely identical to EQT II the most studied family member.UcT I is a strongly basic protein (I.p. 9) with potent hemo-lytic activity. Experiments with lipid monolayers revealedthat UcT 1 requires a mixture of SM:DOPC in order to insertitself into the membrane.

Conclusion: New cytolysin from U. crassicornis sharecommon functional characteristics to proteins belonging toactinoporin family and share a large degree of identity andhomology to EqT II.

Keywords: sea anemone, toxin, cytolysin, actinoporin10.1016/j.toxicon.2012.04.105

105. Discovery, Characterization, and FunctionalImplications of Conotoxins from Cone Snails Species ofthe Americas

Aldo Franco 1, Mari Heighinian 1, Monica Mejia 1,Jessica McCall 1, Shiva Nag 2, Kalyana Akondi 3,Christian Melaun 1,2,3, Norelle Daly 3, Charles W. Luetje 1,2,3,Paul F. Alewood 3, David J. Craik 3, Tanja Godenschwege 1,David J. Adams 2, Frank Marí 11Department of Chemistry & Biochemistry and Biology, Florida AtlanticUniversity, Boca Raton, Florida, USA2Health Innovations Research Institute, RMIT University, Bundoora, VIC,Australia3 IMB, University of Queensland, Brisbane, QLD, AustraliaE-mail address: [email protected] (F. Marí).

Background: Cone snails are among the most prolificand versatile peptide engineers known. Their venom isa complex concoction composed of modified peptides(conopeptides) that elicit awide range of neurophysiologicalresponses. The biochemical strategy developed by conesnails to target the multiplicity of neuronal receptors hasprovided us with a natural library of toxins with greatpotential therapeutic uses, including the first FDA-approveddrug of marine origin, PrialtTM. The expression of con-opeptides is species-specific, with significant intraspeciesand intraspecimen variations. Accordingly, more than 2,000conopeptides/species can be expressed yielding an enor-mous library of bioactive compounds.We have concentratedour research efforts in exploring the venom of the 200þConus species of that inhabit the waters of the Americas(Western Atlantic and Eastern Pacific regions, respectively).

Methods: Here we described the discovery of con-opeptides by using either biochemical-based approaches orefficacious bioassay-guided methods. Specifically, we haveused SE and RP-HPLC to isolate nanomolar quantities ofnovel conopeptides, such as bru1a, bru3a, bru9a and RegIIA.Their sequences were determined by Edman degradation.Testing of these conotoxins included two-electrode voltageclamp recording on nAChRs subtypes expressed in Xenopuslaevis oocytes. Complementary to this approach, we use invivo electrophysiological measurements to evaluate theeffect of conopeptides fractions on the functional outputs ofa well-characterized neuronal system in Drosophila mela-nogaster known as the giant fiber circuit (GFC).

Results: These new a-conotoxins have unique selec-tivity profiles; i.e., RegIIA is a potent inhibitor of a3b4nAChRs, without blocking the a4b2 subtype. This featuremakes RegIIA a suitable probe to evaluate the machineryinvolved in nicotine addiction. While structurally relatedto other a-conotoxins, RegIIA has an exquisite balance ofshape, charges, and polarity exposed on its structure thatenable inhibition of a3b4 nAChRs. A novel a4/3 conotoxin,bru1b, was discovered using GFC approach, and it wasfurther characterized using voltage clamp measurementson a panel of nAChRs subtypes expressed in Xenopusoocytes. Additionally, we will describe a new set of mini-Mconotoxins of the m1-m3 subclasses. These conotoxinsexhibit a remarkable level of structural diversity in theirposttranslational modifications, size of intercystine loopsand overall 3D structure. One of these mini-M conotoxins,bru3a, is active in the GFC indicating a potential target forthese conotoxins.

Conclusions: These novel conopeptides add furthermolecular diversity to the Conus biochemical strategy forpredation and are of particular interest as shown to havenovel functional implications within known conotoxinsuperfamilies.

Keywords: conotoxins, nAChRs, oocytes, neuronal circuits, ion channels10.1016/j.toxicon.2012.04.106

106. Molecular Diversity of Box Jellyfish Toxins

Diane L. Brinkman 1, Jason Mulvenna 2,Nicki Konstantakopoulos 3, Wayne C. Hodgson 3,Geoffrey K. Isbister 3, Jamie E. Seymour 4, James N. Burnell 51Australian Institute of Marine Science, Queensland, Australia2Queensland Tropical Health Alliance, James Cook University, Queensland,Australia3Monash Venom Unit, Dept of Pharmacology, Monash University, Melbourne,Victoria, Australia4 School of Marine and Tropical Biology, James Cook University, Queensland,Australia5 School of Pharmacy and Molecular Sciences, James Cook University,Queensland, AustraliaE-mail address: [email protected] (D.L. Brinkman).

Review: Box jellyfish (cubozoans) are renowned for theirability to immobilise and kill prey and inflict painful anddebilitating stings to humans by injecting potent venomsfromtheirnematocysts.Chironexfleckeri is the largest speciesof box jellyfish and its venom produces extremely rapid andpotentially life-threatening effects. Advances in box jellyfishtoxinology using bioactivity-guided purification methods,tandem mass spectrometry and molecular cloning tech-niques have revealed thatC.fleckeri venomcontains a diversearray of proteins that is dominated by a family of abundanthigh molecular weight venom proteins that are cytolytic,cytotoxic and cause profound cardiovascular collapse inexperimental animals. Related toxins with similar biologicalactivities are present in other jellyfish species and compar-ative analysis of available toxin sequences infers that thisexpanding family of potent cnidarian toxins forms at leasttwo distinct protein clades. Sequence divergence amongfamily members coupled with experimental evidencesuggests there are significant structural variations between




clades that may alter their function and target specificity. Inthis context, an overview of this unique family of proteintoxins ispresented, including abrief historyof theirdiscoveryand recent progress in their purification and molecularcharacterisation primarily from a bioinformatic perspective.

Keywords: bioinformatics, Cnidaria, Cubozoa, cytolysin, cytotoxin, nema-tocyst, venom, toxin10.1016/j.toxicon.2012.04.107

107. The Chemical Landscape of Cnidarians as ViewedThrough the Lens of Pore-Forming Proteins

Tamar Rachamim1,2, Hen Kestenboim 3, Amir Zlotkin 3,Eliahu Zlotkin 2,4, Daniel Sher 1,21Department of Marine Biology, Leon H. Charney School of Marine Sciences,University of Haifa, Israel2Department of Cell and Animal Biology, The Hebrew University ofJerusalem, Israel3Biofouling Research Lab, Hutchinson Water Israel, Tel-Aviv, Israel4Deceased May 18, 2008E-mail address: [email protected] (D. Sher).

Background: Cnidarians such as hydra, sea anemones,corals and jellyfish are simple, mostly sessile animals thatdepend on bioactive chemicals for survival. Cnidariansutilize sophisticated stinging cells (nematocytes) to injectparalyzing venom into their prey, predators or competitors.In addition to the nematocyte venom, we show here thatcnidarians produce cytolytic, “toxin-like” pore-formingproteins (PFPs) in other tissues, and suggest functionalroles for these proteins.

Results and Discussion: Equinatoxins (Eqts), well-studied lethal PFPs from the sea anemone Actinia equina, aredetected by immunohistochemistry in nematocytes and arethus probably used for prey capture. However, Eqts are alsosecreted into the mucous layer surrounding the anemonefrom gland-like cells. Eqt-2 can kill fish upon application tothe water in which they swim, suggesting it may serve fordefense against predators. Surprisingly, while Eqt-2 does notkill bacteria it strongly inhibits their attachment to substrates,suggesting this protein may inhibit the adhesion of patho-genic bacteria or other fouling organisms to the anemone. Asecond example of non-nematocystic PFPs are Hydralysins(Hlns), neurotoxic PFPs from the green hydra Chlorohydraviridissima: in-situ hybridization and immunohistochemistryreveal thatHlns are secreted into the gut cavity upon feeding,where they may promote osmotic disintegration of the preyduring feeding. Other potential PFPs may be involved indevelopmental signaling and immunity in hydra.

Conclusions: We propose a model whereby, in cnidar-ians, bioactive compounds such as PFPs are secreted bothas localized point sources (nematocyte discharges) andacross extensive body surfaces, likely combining to createcomplex “chemical landscapes”. We speculate that thesecnidarian-derived chemical landscapes may affect thesurrounding community on scales from microns to, in thecase of coral reefs, hundreds of kilometers.

Keywords: Cnidaria, hydra, nematocyst, pore-forming protein, chemicallandscape, diffusion, boundarylayer, antimicrobial10.1016/j.toxicon.2012.04.108

108. Cardiovascular and Hemolytic Effects of Sp-CTxa Cytolysin Isolated from the Scorpionfish, Scorpaenaplumieri

Helena L. Gomes 1, D. Freire Davi Jr. 1, Filipe Andrich 1,Edna F. de Medeiros 2, Jader Cruz 3,Antonio N.S. Gondim3,4, Dalton V. Vassalo 1,Suely G. Figueiredo 1

1Universidade Federal do Espírito Santo, Departamento de CiênciasFisiológicas, Vitória, ES, Brazil2Universidade Federal do Espírito Santo, Departamento de Química, Vitória,ES, Brazil3Universidade Federal de Minas Gerais, Departamento de Bioquímica eImunologia, Belo Horizonte, MG, Brazil4Universidade do Estado da Bahia, Departamento de Educação – Campus XII,Guanambi, BA, BrazilE-mail address: [email protected] (H.L. Gomes).

Background: In a previous study, a potent hemolytic/cardiotoxin (Sp-CTx) was isolated from scorpion fish Scor-paena plumieri venom. In the present work we aimed tooptimize the Sp-CTx purification method and to investigatethe mechanisms responsible for its pharmacological effects.

Methods: Sp-CTx was purified to homogeneity fromfish fin spines venom through a combination of ammoniumsulfate precipitation and two chromatographic steps:hydrophobic interaction and anion exchange. Active frac-tions were identified by hemolytic assay. Osmotic protec-tion assays using polyethylene glycol polymers of varioussizes (30mM) were performed to test whether Sp-CTxinduces hemolysis by membrane pore formation. Cardio-vascular studies of Sp-CTx were evaluated on male Wistarrats both in vivo, and in vitro. The effects of Sp-CTxon L-typeCa2þ current (ICa,L) in adult rat ventricular cells wereinvestigated using the whole cell patch clamp method.

Results:Theyieldof thepurificationprocedurewas13%ofthe hemolytic activity from the whole venom, with a purifi-cation factor of 24 fold. Sp-CTx induced-hemolysis decreasedwith increasing size of osmotic protectants. In bolus injectionof Sp-CTx in rats (70mg/Kg) produced a biphasic responsewhich consisted of an initial systolic and diastolic pressureincrease followed by a sustained decrease of both parame-ters. In isolated papillary muscle, Sp-CTx (80nM) producedan increase in isometric force. At 8nM concentration, thistoxin increased by about 30% the ICa,L in rat ventricular cells.

Discussion: In the present work, a new purificationprocedure of Sp-CTx was established. This method requiresa smaller number of chromatographic steps and the activityrecovery was improved substantially (13 fold). Hemolyticeffect was prevented by the presence of osmotic protec-tants. At 40 nM concentration, 100% cell lysis was observedafter 6 min, molecules larger than 3 nm in diameterinhibited 100% cell lysis. As we observed a biphasicresponse in pressure levels when Sp-CTx was injected inrats we could argue that Sp-CTx targets first calciumchannels and after some time non-selective pore formationtakes place leading to a decrease in both systolic and dia-stolic pressure levels. On the other hand, the increase incontraction force would be the result of ICa,L increase.

Conclusion: The results strongly suggest that Sp-CTxmay be a pore-forming protein. These data are in agree-ment with the significant hemolytic activity observed. Also,




the increase in ICa,L paralled by the pore formation mayprovide the basis for Sp-CTx cardiotoxic effects.

Financial support: CNPq, INCTTOX, Toxinologia/CAPES,FACITEC, FAPES.

Keywords: Scorpaena plumieri, cardiotoxin, cytolysin10.1016/j.toxicon.2012.04.109

109. A Comparison of the Structural Characteristics ofthe Nematocysts of the “Fire Corals”Millepora alcicornisand M. complanata, and their Hemolytic andVasoconstrictor Effects

Alejandro García-Arredondo 1, Alejandra Rojas 1,César Ibarra-Alvarado 1, Roberto Iglesias-Prieto 2

1Departament of Chemical and Pharmacological Natural Products Research,Facultad de Química, Universidad Autónoma de Querétaro, Mexico2 Instituto de Ciencias del Mar y Limnología, Universidad Nacional Autónomade MéxicoE-mail address: [email protected] (A. García-Arredondo).

Review:Millepora species are colonial polyp cnidarians ofthe class Hydrozoa that produce calcium carbonate skele-tons. These species are commonly known as “fire corals”because a brief contact with them causes intense burningpain, erythema, wheals, and sometimes ulceronecroticlesions. These local lesions are due to the presence ofstinging organelles named nematocysts, which inject anunidentified mixture of toxins through the skin. Up to now,13 species of the genus Millepora are recognized around theworld. However, it is not clear whether these speciesproduce different type of toxins each from other and if thesame type of toxins are present in the same type of nema-tocyst. Since, in the present study we made a comparisonbetween the types of nematocysts present in two Milleporaspecies that inhabit in shallow-water reefs of the MexicanCaribbean:M. alcicornis andM. complanata. In this study, wealso the differences in profile proteins of their extracts andthe vasoconstrictor and hemolytic activities. First, the type ofnematocysts of both species was examined using lightmicroscopy, as well as scanning and transmission electronmicroscopy. Only two types of nematocysts were observedin both species. The most abundant type was identified asmacrobasic mastigophore and the other one belonged to thestenotele type. Second, both species induce the two types ofactivities, but the vasoconstrictor effect induced by theextract ofM. alcicornis (Emax¼ 90.1�5.6 % of the contractioninduced by 1 mM phenylephrine) was slightly more efficientthan that induced by the extract of M. complanata (Emax ¼64.6� 2.7 %), although there is not a significant difference inthe potency. In the other hand, analysis of the hemolyticactivities showed that M. alcicornis extract (HU50 ¼ 0.05 �0.005 mg protein/ml) was significantly 10-fold more potentthan the extract of M. complanata (HU50 ¼ 0.5 � 0.02 mgprotein/ml). In conclusion, the results of the present studyshown that althoughM. alcicornis andM. complanta presentsthe same types of nematocysts, there are some differences intheir protein profiles and in the potency of their extracts toinduce hemolysis. These observations suggest that the sametype of nematocyst can produce different types of toxins andin variable quantities.

Financial support: This work was supported by grantCB-2009-01 (Project 133785) to Alejandra Rojas from theMexican Council of Science and Technology (CONACYT).

Keywords: Millepora complanta, Millepora alcicornis and Nematicysts.10.1016/j.toxicon.2012.04.110

110. Voltage Sensor Trapping in Voltage-Gated K-Channels by the Marine Neurotoxin Gambierol

Ivan Kopljar 1, Alain J. Labro 1, Jon D. Rainier 2, Jan Tytgat 3,Dirk J. Snyders 1

1University of Antwerp, Dept. of Biomedical Sciences, Antwerp, Belgium2University of Utah, Dept. of Chemistry, Salt Lake City, UT, USA3University of Leuven, Laboratory for Toxicology, Leuven BelgiumE-mail address: [email protected] (D.J. Snyders).

Background: We previously showed that the polyetherladder toxin gambierol (a ciguatera toxin) acts on a novelbinding site at the lipid exposed faceof theporedomainofKvchannels.

Methods: Voltage clamp recordings of ionic currentsand gating currents were obtained from HEK293 cellsexpressing Kv3.1 channel subunits (WT and insensitivemutants). Concatemers withWTandmutant subunits wereused to constrain subunit stoichiometry. ComputationalMarkov models with appropriate gating and drug bindingschemes were implemented to investigate the gatingmodification.

Results: Gambierol inhibited ionic current throughKv3.1 channels with an IC50 of 1-2 nM, with a time constantofw 60 secwhen the channels were held in the closed stateat -80 mV. Short (250 ms) steps to þ140 mV failed toovercome this inhibition, but 29% of ionic current wasrecovered after prolonged (5-10 sec) depolarizations toþ140 mV, suggesting a lower affinity of the open state. Thiswas further tested by keeping the cells depolarized at þ60mV while washing in the toxin; under these conditions nonoticeable block developed. The gating currents of Kv3.1channels displayed “OFF” currents with a slowed currentdecay after steps that open the channel gate, indicating thatthe concerted opening step has been passed (similar toShaker channels). After application of Gambierol, thisslowing quickly disappeared and subsequently, all gatingcharge movement was eliminated. A tetrameric con-catemer with only 1 high-affinity binding site still dis-played high toxin sensitivity, but displayed faster unblockat strong depolarizations (>120mV).

Discussion: Given the tetrameric structure of Kvchannels, there are 4 binding sites for gambierol on thelipid exposed crevice between S5 and S6. The resultsindicate that binding of gambierol prevents to thevoltage sensors to move to the activated position, bystabilizing a deep closed state, such that binding ata single binding site is sufficient to preclude channelopening (which requires all 4 voltage sensors to reachthe activated state). Computational modeling confirmedthis mechanism of ultra-strong stabilization of the closedstate.

Conclusions: The polyether toxin gambierol acts asa profound gating modifier at a lipid exposed binding site




and with a mechanism that is distinct from the gatingmodification of hanatoxin-related toxins.

Keywords: Kv channels, gating modification, gambierol, ciguatera10.1016/j.toxicon.2012.04.111

111. State of the Art of Palytoxin and Analogs AnalyticalMethods for Seafood Monitoring

Vitor Vasconcelos 1,2, Joana Azevedo 2,3, Marisa Silva 1,2,Vitor Ramos 1,2

1Department of Biology, Faculty of Sciences, Porto University, Portugal2Marine and Environmental Research Center – CIIMAR/CIMAR PortoUniversity, Portugal3 School of Health Technology of Porto, Vila Nova de Gaia, PortugalE-mail address: [email protected] (V. Vasconcelos).

Review: Palytoxin group (PLTXs) are produced bymarineorganisms, like zoanthids (Palythoa) and benthic dinofla-gellates (Ostreopsis). Besides originally found in tropicalregions, reports on the presence of PLTXs in subtropical andtemperate marine waters have grown considerably in thepast decade. This is related to the increase of Ostreopsis spp.blooms in such environments, which pose considerable riskto the human health. In fact, contamination of seafood withOstreopsis spp. already caused human poisoning incidentsafter the ingestion of clupeoid fish, somewith fatal outcomes[1,2]. Therefore there is an urgent need to establish regula-tions on PLTX-group toxins in seafood intended for humanconsumption. Due to lack of exposure assessment andanalytical information about these toxins, authorities are notable to establish a reliable guideline limit. European FoodSafety Authority (EFSA) gives an estimated value for paly-toxin and ostreocin-D of 30 mg/Kg shellfish meat [1]. Chem-ically these natural products are large and complexmolecules, having both hydrophilic and lipophilic behaviorsconsisting of 64 chiral centers, 40-42 hydroxyl and 2 amidegroups. These characteristics confer their heat resistance,water solubility and a large number of possible analogs. Theanalytical approach to identify and quantify PLTXs, such asHPCE-UV, HPLC-UV and FLD or LC-MS, it's mainly chosendependingon the type ofmatrix and on the limit of detectionrequired. The most sensitive method for Ostreopsis cultures,having a limit of detection (LOD) of 0.75 ng on column [3,4],implies the derivatization of the molecule and untilknow was not validated for seafood intended for humanconsumption. In order to better monitor this emergentphenomenon there is a need to develop techniques and toproduce certificated standards and reference materials toachieve proper method validation, following InternationalConference on Harmonization (ICH) and Food andDrug Administration (FDA) guidelines. This work overviewsthe actually available methods to analyze PLTXs - high-lighting their strengths, limitations and adequacy for seafoodmonitoring, while it points out possible analytical require-ments and future approaches.

References

EFSA Panel on Contaminants in the Food Chain (CONTAM),2009. The EFSA Journal 1306, 1–23. Available from: www.efsa.europa.eu (December 2010).

Ramos, V., Vasconcelos, V., 2010. Mar. Drugs 8, 2021–2037.Rióbo, P., Paz, B., V., Franco J. M., 2006. Anal. Chim. Acta 566,217–223.Rióbo, P., Franco J. M., 2011. Toxicon. 57, 368–375.

Financial Support: This project is partially financed bythe interreg project PHARMATLANTIC and the FCT projectPTDC/MAR/099642/2008.

Keywords: palytoxin, analytical methods, seafood monitoring10.1016/j.toxicon.2012.04.112

112. Cathepsin B/X is Secreted by Echinometra lucunterSea Urchin Spines, a Structure Rich in Granular Cellsand Toxins

Juliana M. Sciani 1,2, Marta M. Antoniazzi 3, Carlos Jared 3,Daniel C. Pimenta 1,2

1 Laboratório de Bioquímica e Biofísica, Instituto Butantan, São Paulo, SP,Brazil2Centro de Biologia Marinha, Universidade de São Paulo, São Sebastião, SP,Brazil3 Laboratório de Biologia Celular, Instituto Butantan, São Paulo, SP, BrazilE-mail address: [email protected] (J.M. Sciani).

Background: Echinometra lucunter is a commonBrazilian sea urchin; however, few data is available on itsspines’ structures or toxins. In insects and mollusks thepresence of cathepsins has been described. These enzymeswould aid in the eggs or larva's growth through proteindegradation, and remodeling the calcified part, asCathepsin K does in mammals bones. Here, we reporta cathepsin B/X like activity from the sea urchin spines.

Methods: Spines aqueous extract was obtained in 100mM NH4COOH (pH 7.4) at 4�C and was assayed for enzy-matic activity, with synthetic substrates, in the presence orabsence of inhibitors and DTT. For properly classification ofthe peptidase in the correspondent clan and family, thereference FRET-substrate cleavage pattern was determined,pH-dependency activity was evaluated and Western-blotanalyses were performed. Moreover, spines were analyzedby light and scanning electron microscopy (SEM).

Results: The spine extract was able to cleave both Z-R-MCA and Abz-GIVRAK(Dnp)-OH following pre-incubationwith DTT, and was inhibited by E-64. Furthermore, thedouble-peaked pH curve (5 and 7) and the cleavage siteproportion (4:6, RYA:AYK), indicate the presence of bothmono and dicarboxypeptidase activities. Finally, it waspositive for the (human) anti-cathepsin B antibody in theWestern-blot. According to spines histological analyses, thepresence of granulous cells, positively stained with bro-mophenol blue, along the entire spine, but concentrated atthe tip, were observed. These cells are located within thecalcified matrix, which is longitudinally disposed andradiates outwards, as observed by SEM.

Discussion: The cleavage pattern of FRET substrate, theinhibition by E-64 and DTT-activation clearly indicates thepresence of a cysteine peptidase in the spine extract. Thetwo optimum pH values are typical for some cathepsins, aswell as the carboxy mono and dipeptidyl-peptidaseactivity, characteristic of cathepsin X and B, respectively.


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Moreover, the Western blot analysis confirmed the pres-ence of cathepsin B, among other proteins revealed in thesilver-stained SDS-PAGE. Proteins are normally abundant inthe granular cells, just like those observed in the bromo-phenol blue staining that were more abundant at the tip ofthe spine, a region that can be easily broken due to contact,which would need frequent remodeling.

Conclusion: E. lucunter spines extracts showed a proteo-lytic activity that, based on the selected experiments, couldbe classified as Cathepsin B and/or X, according to theircleavage site. This enzyme could also participate in theremodeling process and growth of the spine.

Fig. 1. Sea Urchin.

Keywords: sea urchin, Echinometra lucunter, spines, cathepsin, granularcells10.1016/j.toxicon.2012.04.113

113. Effects of Successive Subculture and VariousCulture Conditions on Growth and PSP Productivityof the Toxic Dinoflagellate Alexandrium catenella

Shanshan Jiang 1, Sho Miyata 2, Tomohiro Takatani 1,Osamu Arakawa 1

1Graduate School of Fisheries Science and Environmental Studies, NagasakiUniversity, Nagasaki, Japan2Graduate School of Science and Technology, Nagasaki University, Nagasaki,JapanE-mail address: [email protected] (S. Miyata).

Background: Production of paralytic shellfish poison(PSP) in toxic dinoflagellates considerably differs amongthe same species, depending on the strain, habitat, andseason, and even betweenwild cells and cultured cells. Weinvestigated the effects of successive subculture andvarious environmental factors on growth and toxinproduction by the PSP-producing dinoflagellate Alexan-drium catenella (Ac), which frequently cause PSP contam-ination in bivalves in Kyushu, Japan.

Methods: [Exp 1] Wild cells (Wc) of Ac were collectedfrom Tamanoura Bay, Fukue Island, Kyushu, in April2009, and Ac cells isolated from the same seawaterwere successively subcultured in SWM-III medium at 20�Cwith the light intensity of 60 mmol/m2/s (12L/12D), and

primary, secondary, and tertiary subculture cells (Sc-1,Sc-2, and Sc-3, respectively) were obtained. Toxin profilesof each cell group were analyzed by high performanceliquid chromatography with fluorometric detection(HPLC-FLD). [Exp 2] Ac cells were cultured in SWM-IIImedium and trace metal-free SWM-III medium under thesame conditions as in Exp 1 for 16 days. Cell numbers werecounted periodically during the culture, and toxinprofiles at the end of culture were analyzed by HPLC-FLD.[Exp 3] Ac cells were cultured under a red LED (RL; 660nm), blue LED (BL; 470 nm), and white fluorescent light(WFL) for 15 days, and the growth and toxin profiles wereevaluated.

Results and Conclusions: Toxin amounts producedby Wc, Sc-1, Sc-2, and Sc-3 were 646, 50, 12, and 15fmol/cell, respectively. Gonyautoxin (GTX)2,3, GTX6 andsaxitoxins (STXs) disappeared during successive subcul-ture, simplifying the toxin profile; C toxin (C)1-4, GTX1,4,and GTX5. No obvious difference in Ac growth wasdetected when trace metals were excluded, but toxinlevels were more than double those in the controlculture. C1,2 and GTX1,4 levels increased remarkably.Growth of Ac was slow under RL, and maximum celldensity was 1/3 and 1/4 that under BL and WFL,respectively. Ac, however, produced a greater amount oftoxin under RL, 3.7 to 9.3 times higher than under BL andWFL. Under RL, the ratio of C1,2 was higher (about 60%),whereas ratios of GTX1,4 and C3,4 were lower. Theseresults indicate that successive subculture leads toa decrease in Ac production of PSP, and that productionis affected by trace metals and irradiation wavelengthduring culture.

Keywords: paralytic shellfish poison (PSP), saxitoxin (STX), gonyautoxin(GTX), dinoflagellate, Alexandrium catenella10.1016/j.toxicon.2012.04.114

114. Effect of Byproducts from Artemia salina CultureMedium on PSP Toxicity and Toxin Composition ofAlexandrium catenella

Toshio Saito 1, Tadahiro Kogure 1, Tsuyosi Sagara 2,Kedarnath Mahapatra 1, Sachio Nishio 2

1 School of Marine Science and Technology, Tokai University, Shizuoka, Japan2 Shikoku Junior College, Shikoku University, Tokushima, JapanE-mail address: [email protected] (T. Saito).

Background: Presently, it is not clearly known whatcauses the quantitative and compositional change ofparalytic shellfish poisoning (PSP) toxin in the PSP-producing phytoplankton. Most of the investigationscarried out so far used various physical and chemicalparameters such as temperature, salinity, pH, dissolvednutrient ratio etc. for deciphering their possibleinfluence on the PSP toxicity change. However, it'spossible to assume that inhibition of competing co-occurring plankton species could influence the toxinproduction dynamics in marine phytoplankton undernatural condition. In order to understand the influence ofother plankton species on the PSP toxicity and composi-tion, we compared PSP produced by Alexandrium catenella




by adding the rearing medium of Artemia (Artemia salina)to the culture medium vis-à-vis the control medium inwhich filtered natural seawater was added to culturemedium.

Materials and Methods: After rearing for 10 days, theArtemia-rearing medium was filtered through 0.22 mmfilters and added to the in sterile enriched seawater medium(ESM) in three culture flasks. In three separate culture flasks,filtered natural seawater was added to the ESM to use ascontrols. A. catenellawas cultured in both set of medium for6 days under light level of 49,000 lux, and 14h light/10h darkcycle at the temperature of 21�C. After completion of theculture experiment, A. catenella were collected from eachsample for counting cell numbers under a microscope andPSP was extracted from each sample to measure the toxicitylevel and the toxin composition using high performanceliquid chromatography (HPLC). The cell counts and toxinmeasurements were carried out prior to beginning of theexperiments in all the samples.

Results and Discussion: Significant difference (p<0.05)was observed in toxicity/per cell values between the samplewith the Artemia-rearing medium (88.38�23.30 fmol/cell)and the control medium (38.45�20.44 88.38�23.30 fmol/cell). Concerning the PSP composition, there was signifi-cantly higher (p<0.05) concentration of PX2 and GTX4 in thesample with Artemia-rearing medium compared to that ofthe controls. Similar trend was also observed with PX2 andGTX4 toxicity in the sample with the Artemia-rearingmedium compared to that of the controls. The results wereconfirmed by undertaking experiments twice and similarresults were obtained both the times.

Conclusions: Our results suggest that the metabolicsubstances released in the byproducts from biologicalactivity of other plankton could potentially influence toxicityand composition of toxins in A. catenella. Ecological signifi-cance of such phenomena needs to be further investigated.

Keywords: PSP, Alexandrium catenella, toxicity, toxin composition10.1016/j.toxicon.2012.04.115

115. Two Proteins Homologous to Pufferfish Saxitoxin-and Tetrodotoxin-Binding Protein (PSTBP) Found inthe Plasma of Non-Toxic Cultured Specimens of thePufferfish (Takifugu rubripes)

Ryohei Tatsuno 1, Kenichi Yamaguchi 2,Tomohiro Takatani 2, Osamu Arakawa 2

1Graduate school of Science and Technology, Nagasaki university, Nagasaki,Japan2Graduate school of Fisheries Science and Environmental Studies, Nagasakiuniversity, Nagasaki, JapanE-mail address: [email protected] (K. Yamaguchi).

Background: Marine pufferfish generally possess thepotent neurotoxin tetrodotoxin (TTX). Although previousTTX administration experiments using non-toxic culturedspecimens of Takifugu rubripes have provided essentialinformation on the transfer/accumulation profiles of TTXin the pufferfish body, the molecular mechanism ofTTX transportation/accumulation remains unclear. It wasrecently reported that T. rubripes plasma containsw120-kDa

protein that immunocrossreacts with pufferfish saxitoxin-and tetrodotoxin-binding protein (PSTBP) isolated fromTakifugu pardalis. In the present study, we explored PSTBPhomologous genes in T. rubripes, a suitable model species forpost-genomic research, and investigated the expression ofPSTBP homologs in non-toxic cultured specimens.

Methods: Using the T. pardalis PSTBP as the querysequence, a BLAST search was conducted against a draftgenome sequence of T. rubripes. 3’-RACE was performed toobtain complete coding sequences. Multiple sequencealignment was performed using CLUSTAL W/X, and phylo-genetic analysis, using the neighbor-joining method withthe PAUP program. Expression of mRNAs in various tissues(skin, muscle, liver, testis, and ovary) was examined by RT-PCR. A 120-kDa protein was separated by SDS-PAGE afterammonium sulfate fractionation of the plasma, and iden-tified by N-terminal protein sequencing (Edman chemistry)and MALDI-QIT-TOF mass spectrometry.

Results: Three putative genes (tentatively designatedTr1-3) were obtained by the BLAST search/3’-RACE. Tr1 andTr2 genes appeared to be novel, encoding a single lipocalindomain, which is closely related to the C-terminal domainof T. pardalis PSTBP. The Tr3 gene could be a functionalcounterpart of PSTBP, possessing two lipocalin domains.The phylogenetic tree indicated that lipocalin domains of Trproteins and T. pardalis PSTBP form three distinct clades.Tr1 and Tr3 gene transcripts were detected exclusively inthe liver. The 120-kDa protein from the plasma was iden-tified as the mixture of Tr1 and Tr3.

Discussion: Tr1 protein may form an SDS-resistantdimer, as Tr1 (single-domain) was co-identified with Tr3(two-domain) in the same protein band (120 kDa). Thepossibility that Tr1 is the product of an unidentified genethat encodes tandem-repeated Tr1-like domains, however,cannot be ruled out. Constitutive expression of Tr1 (or Tr1-like) and Tr3 in non-toxic pufferfish would be beneficial forquick self-protection from free toxins upon toxin-intake,and also for efficient toxin transportation/accumulation.Tr2 gene expression is currently under investigation.

Keywords: pufferfish, Takifugu rubripes, tetrodotoxin, toxin-bindingprotein, PSTBP, homologous gene, MALDI mass spectrometry10.1016/j.toxicon.2012.04.116

116. Systemic Toxicity of the “Fire Coral” Milleporacomplanata: Isolation of a Non-Protein VasoconstrictorFraction with Lethal Activity in Mice

Alejandro García-Arredondo 1, Alejandra Rojas 1,César Ibarra-Alvarado 1, Moustapha Bah 2

1Departament of Chemical and Pharmacological Natural Products Research,Facultad de Química, Universidad Autónoma de Querétaro, Querétaro,Querétaro, México2CEACA, Facultad de Química, Universidad Autónoma de Querétaro.Querétaro, Querétaro, MéxicoE-mail address: [email protected] (A. García-Arredondo).

Background: M. complanata is a cnidarian (classHydrozoa) widely distributed in the coral reefs of theMexican Caribbean. This hydrocoral is popularly known as“fire coral”, since contact with it causes severe pain, skineruptions and blisters. Previous in vitro experiments,




carried out by our research group, showed that the aqueousextract of this hydrocoral contained proteins that inducedseveral pharmacological and toxic effects, which includehemolysis and stimulation of intestinal and arterial smoothmuscle contractility.

Methods: In the present study, the systemic toxicity ofM. complanata aqueous extract was assessed in mice.Additionally, a bioactivity directed chromatographic frac-tionation of the extract was performed and the bioactivefractions were further analyzed in order to isolate thecompounds responsible for the pharmacological and toxi-cological activities induced by the extract.

Results: The results obtained in the present study indi-cated that intravenous administration of the extract inducedviolent convulsions and death in mice within 1 min (LD50 ¼4.62 mg protein/g of body weight). Doses less than the LD50(1.33, 2.67 y 4.00 mg protein/g) produced kidney and lunghistopathological damage attributed to the presence ofcytolysins. Histopathological changes were completelyeliminated after incubation of the extract in denaturingconditions. Unexpectedly, the denatured extract conservedits lethal effect. These findings suggested that that the M.complanata extract contained hemolysins that might beresponsible for the histopathological changes observed inkidney and lung. Additionally, this extract contained otherunidentified thermostable toxins, likely secondary metabo-lites, which have lethal effects in mice. Chromatographicanalysis of the extract led to the isolation of a 61 kDa vaso-constrictor protein. Additionally, several non-peptidic vaso-constrictor compounds were detected. Particularlyinteresting, fraction MC1-IIA, obtained by a three-stepprocess (ion exchange, gel filtration and reverse phasechromatography), induced vasoconstriction, delayed hemo-lysis, and lethal effects in mice. These effects resembledthose produced by the crude extract. A subsequent chro-matographic analysis of MC1-IIA showed that this fractioncontained at least four secondary metabolites, which werenamed as IIA-2-1, IIA-2-2, IIA-3-1, and IIA-3-2. Analysis ofmass spectrometric and NMR spectroscopic data indicatedthat these metabolites were poly-hydroxylated compounds.

Conclusions: The present study constitutes the firstreport of the presence of a non-peptidic lethal toxin in anorganism of the class Hydrozoa, and evidenced the greatstructural diversity of the toxins produced by Milleporaspecies.

Financial support: This work was supported by grantCB-2009-01 (Project 133785) to Alejandra Rojas from theMexican Council of Science and Technology (CONACYT).

Keywords: fire coral, histopathological damage, lethal effect.10.1016/j.toxicon.2012.04.117

117. Identification and Characterization of a-Conotoxinsin Conus purpurascens

Alena M. Rodriguez 1,2, Frank Marí 21 The National Science Foundation, Undergraduate Research and MentoringProgram, USA2 Florida Atlantic University, Department of Chemistry and Biochemistry,Boca Raton, FL USAE-mail address: [email protected] (A.M. Rodriguez).

Background: Cone snails use venom that containproteins and highly modified peptides (conopeptides) torapidly immobilize their prey and defend against predators.Conopeptides act directly upon specific ion channels andreceptors in both neuronal and neuromuscular tissues. Themost ubiquitous family of conopeptides across the Conusgenus is the a-conotoxins. a-conotoxins antagonize nico-tinic acetylcholine receptors (nAChRs). These receptors areassociated with several disease states, such as Alzheimer'sdisease, Parkinson's disease, and nicotine addiction. Conuspurpurascens is the only fish-hunting species of the EasternPacific. The venom of this species is known to containvarying components between individuals, which highlyincrease the number of conopeptides present in C. pur-purascens. Although two a-conotoxins have already beenfully characterized from its venom, there are numerousunidentified a-conotoxins that have yet to be discovered.

Methods: The venom of thirty-seven captive C. pur-purascens individuals was extracted using a “milking”procedure and then separated into individual componentsusing size exclusion chromatography (SEC) and reverse-phase high performance liquid chromatography (RP-HPLC).The molecular weights of the isolated conopeptides weredetermined using matrix assisted laser desorption ioniza-tion mass spectrometry (MALDI-MS). The conopeptideswere plated on twomatrices for MALDI-MS analysis, alpha-cyano-4-hydroxycinnamic acid (a-CHCA) and the partiallyreducing matrix, 1,5-diaminonaphthalene (1,5-DAN).

Results: The recently discovered a-conotoxin, a-PIC,and other conopeptides of novel molecular weights (1422,1560, and 1810 Da) were collected.

Discussion: By comparing the molecular weights ofconopeptides on each matrix, the number of disulfidebonds can be determined, and therefore, the conopeptidecan be classified according to its conotoxin framework. Thenovel conopeptides will be tested in vivo for bioactivity inthe Drosophila melanogaster giant fibre circuit (GFC). TheD. melanogaster GFC contains nAChRs that are homologousto those in vertebrates.

Conclusion: Since there is interspecies variation betweenthe venom of C. purpurascens individuals, additional novelconopeptides with important biological activity can dediscovered. Testing novel conopeptides in theDrosophilaGFCsystem is one of the many ways that we can begin to char-acterize themby their targets. Characterizing a-conotoxins isof great importance as they can be used as pharmaceuticalprobes for disease pathways and neurodegeneration.

Keywords: Conus purpurascens, a-conotoxins, nicotinic acetylcholinereceptors10.1016/j.toxicon.2012.04.118

118. Primary Structures of Proteinaceous Toxins fromThree Species of Scorpaeniform Fish (Lionfish, Pteroislunulata, Scorpionfish, Inimicus japonicus andWaspfish,Hypodytes rubripinnis)

Aya Kiriake, Yasuko Suzuki, Yuji Nagashima, Kazuo ShiomiTokyo University of Marine Science and Technology, Department of FoodScience and Technology, Tokyo, JapanE-mail address: [email protected] (A. Kiriake).




Background: The order Scorpaeniformes includesa number of venomous fish, such as stonefish, lionfish andscorpionfish. These venomous fish possess pungent spineswith venom glands or venom sacs in dorsal, pelvic and analfins. When stung by the spines, local symptoms such asintense pain, swelling and redness are immediately inducedin victims; even deaths may occur in sever cases. So far,stonefish proteinaceous toxins, stonustoxin of Synanceiahorridaandneoverrucotoxin (neoVTX)ofSynanceiaverrucosa,have beenwell characterized and elucidated for their primarystructures. Recently,wedetermined the primary structures oftoxins from two species of Pterois lionfish, P. antennata andP. volitans, based on a cDNA cloning strategy using primersdesigned from the reported amino acid sequences of thestonefish toxins. It is assumed that proteinaceous toxins ofother venomous scorpaeniform fish can be determined fortheir primary structures by the same strategy.

Methods: Three species of scorpaeniform fish (lionfishPterois lunulata of the family Scorpaenidae, scorpionfishInimicus japonicus of the family Synanceiidae and waspfishHypodytes rubripinnis of the family Tetrarogidae) were usedas samples. The crude toxin extracted from dorsal spines ofeach live specimenwas assayed for hemolytic activity againstrabbit erythrocytes and lethal activity against mice andanalyzed for proteins cross-reacting with neoVTX by immu-noblotting and inhibition immunoblotting using the anti-neoVTX antiserum previously raised in a guinea pig. Primarystructures of proteinaceous toxinswere determined by cDNAcloning using primers designed from the highly conservedamino acid sequences of the stonefish and lionfish toxins.

Results and Discussion: All the crude toxins from thethree species of scorpaeniform fish showed both hemolyticand lethal activities with considerably different potencies.Analysis by immunoblotting and inhibition immunoblottingrevealed that the scorpaeniform fish contain about a 75 kDaprotein (corresponding to the toxin subunit) cross-reactingwith neoVTX. Cloning experiments established the aminoacid sequences of the scorpaeniform fish toxins (a andb subunits composed of 698-703 amino acid residues). Theestablished sequences share high identities (more than 80%)with one another and alsowith those of the known stonefishand lionfish toxins. A B30.2/SPRY domain was also recog-nized in the C-terminal region of the scorpaeniform fishtoxins, as in the case of the known stonefish and lionfishtoxins. These results suggest a universal distribution ofbiologically and structurally similar proteinaceous toxins invenomous scorpaeniform fish.

Keywords: primary structure, proteinaceous toxin, scorpaeniform fish10.1016/j.toxicon.2012.04.119

119. Characterization of Local Inflammatory ResponseInduced by Scorpion Fish, Scorpaena plumieri Venom ina Mouse Model of Tissue Injury

Thiago N. Menezes 1, Juliana B.T. Carnielli 1,Pedro H. Lemos 1, Nazaré S. Bissoli 1,Mônica Lopes-Ferreira 2, Filipe Andrich 1,Suely G. Figueiredo 1

1Universidade Federal do Espírito Santo, Departamento de CiênciasFisiológicas, Vitória, ES, Brazil

2 Instituto Butantan, Laboratório Especial de Toxinologia Aplicada(CEPID/FAPESP), São Paulo, SP, BrazilE-mail address: [email protected] (S.G. Figueiredo).

Background: The Scorpaena plumieri fish venom inducesa severe pain and edema, observed both clinically andexperimentally. In order to understand more about theenvenomation syndrome, the present study characterizedexperimentally the local acute inflammatory responseinduced by S. plumieri venom (SpV) in a mouse model oftissue injury.

Methods: Venom local activities (15 mg) were assayedusing mice hind paw method. The local edema was quan-tified by measuring the thickness of injected paws; histo-pathological analysis (leukocyte recruitment andmorphological changes) were assessed in injected foot-pads; cytokines (TNFa and IL-6) and chemokine (MCP-1)levels were measured in paw supernatants homogenatesby flow cytometry using Cytometric Bead Array – MouseInflammation Kit. The mechanism involved in the SpVedematogenic activity was investigated by previous treat-ment of mice (i.p.) with anti-inflammatory drugs:i) cyclooxygenase non-selective inhibitor, diclofenacsodium (1 mg/Kg); ii) histamine H1 receptor antagonist,promethazine (1 mg/Kg); iii) serine-proteases inhibitor,aprotinin (8 mg/Kg) and iv) bradykinin B2 receptor antag-onist, HOE-140 (100 mol/Kg).

Results: SpV induced an intense and sustained dose-dependent local edema. In addition, an expressiveincrease of the number of leukocytes was observed(neutrophil cells were predominant), reaching maximalintensity after 6 h of venom injection. SpV also was ableto induce a significant release of pro-inflammatorymediators. Maximal levels of TNF-a (38 rg/ml), IL-6(1600 rg/ml) and MCP-1(2470 rg/ml) were recordedafter 2 h of venom injection. SpV induced edema wassignificantly reduced by previous administration ofaprotinin or HOE-140. However, the pre-treatment withdiclofenac sodium and promethazine had less effect onthis response.

Discussion: In the present study, we demonstrated thatthe local inflammatory response induced by SpV is char-acterized by fast release of some pivotal proinflammatorycytokines and chemokine, which was accompanied byleukocyte recruitment. The onset of the acute inflammatoryresponse (leukocyte accumulation) was broadly consistentwith release of TNF detected in footpad homogenates0.5 and 2 h after venom administration. The significantreduction of edematogenic response by HOE-140 andaprotinin evidences that one of the main pathwaysinvolved in SpV-induced edema is the kallikrein-kininsystem (KKS).

Conclusion: Our results demonstrate that SpV evokesa complex inflammatory reaction and provide clearevidence of the involvement of the KKS in this response.

Financial support: CNPq, INCTTOX, Toxinologia/CAPES,FACITEC, FAPES.

Keywords: Scorpaena plumieri venom, edema, inflamatory mediators,kallikrein-kinin system10.1016/j.toxicon.2012.04.120



120. The Atypical Activity Profile of bru1b, ana-Conotoxin from the Venom of Conus brunneus

Mari D. Heghinian 1, Monica Mejia 2,Tanja A. Godenschwege 2, Frank Marí 11Departments of Chemistry, Florida Atlantic University, Boca Raton, FL, USA2BiochemistryBiological Science, FloridaAtlanticUniversity, BocaRaton, FL, USAE-mail address: [email protected] (M.D. Heghinian).

Background: Cone snails are venomous marine preda-tors whose venom is a complex mixture of modifiedpeptides (conopeptides). Conopeptides have direct speci-ficity towards voltage- and ligand-gated ion channels andG-protein coupled receptors. More specifically, a-conotoxinstarget nicotinic acetylcholine receptors (nAChR) and are ofgreat interest as probes for different nAChR subtypesinvolved in a broad range of neurological function.

Methods: We used in vivo electrophysiologicalmeasurements of the functional outputs of a well-charac-terized neuronal circuit in Drosophila melanogaster knownas the giant fiber system, a circuit of four neurons inner-vating to two muscles, the TTM and DLM, which areresponsible for the fly's escape response. Additionally,bru1b was tested using voltage clamp of Xenopus oocytesexpressingmammalian nAChRs and in binding assays usingAplysia californica acetylcholine binding protein (AChBP).The binding modes of bru1b with the Da7 nAChR weremodelled using Modeller9.10v and the crystal structures ofthe Aplysia AChBP-conotoxin known to date.

Results: A new a-conotoxin has been identified thatdoes not have the same activity profile as other a-con-otoxins. This conotoxin was originally discovered viabioassay-guided fractionation using the recently developedDrosophila melanogaster Giant Fiber circuit assay1. Afterbiochemical characterization, it was determined that thisconotoxin was an a-4/3 conotoxin that specifically blockedthe DLMn neuronal pathway of the giant fiber system at 33pmol/mg. It was tested against most subtypes of mamma-lian nAChRs, and found to be inactive at the micromolarrange. It was also tested against the Aplysia californicaAChBP and was found not to bind to it.

Discussion:Theatypical activityprofileofbru1bwasquiteunexpected. This conotoxin is an insect-specific Da7-nAChRinhibitor. To date, no D. melanogaster nAChR subunits havebeen expressed in any other expression systems.

Conclusion: The model of bru1b's binding modewith the Da7 nAChR based on the crystal structure ofthe Aplysia californica AChBP provides an explanation ofbru1b's interesting activity profile and shows yet anothervery selective tactic of cone snail venom used to captureand immobilize their prey.

Keywords: conotoxin, drosophila, nicotinic acetylcholine receptor10.1016/j.toxicon.2012.04.121

121. Venom Composition Changes during Prey Capturein Conus textile

Cecilia Prator, Joseph SchulzDepartment of Biology, Occidental College, Los Angeles, CA, USAE-mail address: [email protected] (J. Schulz).

Background: We investigated venom compositionvariation during prey capture in the mollusc-huntingConus textile. While studies on injected venom havebecome more common in fish-hunting cone snails, theseapproaches have not been applied to the analysis ofvenoms from mollusc-hunters. Observations of themollusc-hunter C. textile during feeding reveal that preyare often injected multiple times in succession. Speciesthat inject prey multiple times during a single feedingevent may have compositional changes in their injectedvenom profiles. Our work investigates the occurrence ofvenom composition changes after each injection and howthese changes are related to the biomechanics of prey-capture.

Methods: C. textile injected venom was collectedfrom multiple shots in succession. Injected venom samplesfrom multiple individuals were analyzed by mass spectrom-etry and reverse-phase high performance liquidchromatography.

Results: We have found novel peptides not previouslydescribed from studies on dissected venom. Initial resultssupport intraspecific venom variation as well as complexvenom profiles that show compositional differences insubsequent venom injections during a single feeding event.

Discussion: Venom profiles from a mollusc-hunterreveal venom profiles to be more complex than profilesobtained by fish-hunting cone snails. The results obtainedby MADLI-ToF MS are mirrored by quantitative differencesin venom composition shown by reverse-phase highperformance liquid chromatography.

Conclusions: It is not yet clear why mollusc-huntersinject prey multiple times prior to engulfment of preyand how this relates to the observed changes in venomcomposition. Analysis of injected venom from multipleC. textile individuals provides some insights towardunderstanding this phenomenon. Future studies on thephysiological effects and molecular targets of thevenom peptides will help reveal the role of the variousvenom peptides injected during first versus laterinjections.

Keywords: cone snail, venom, prey capture10.1016/j.toxicon.2012.04.122

122. Acute Toxicity and Brine Shrimp CytotoxicityInduced by the Venom of the Fire Coral M. alcicornisCollected in the Mexican Caribbean

Rosalina Hernández-Matehuala 1,2,Alma A. Vuelvas-Solórzano 1,2,Armando Zepeda-Rodríguez 3, Lurdes Palma 4,Alejandra Rojas 2

1 Posgrado en Ciencias del Mar y Limnología, Universidad NacionalAutónoma de México 04510, México D. F., México2 Laboratorio de Investigación Química y Farmacológica de ProductosNaturales, Facultad de Química, Universidad Autónoma de Querétaro,Querétaro, Qro., México3Departamento de Fisiología celular y de tejidos, Escuela de Medicina,Universidad Nacional Autónoma de México, Ciudad de México, México4Unidad de Microscopía, Instituto de Neurobiología, Universidad NacionalAutónoma de México, Querétaro, MéxicoE-mail address: [email protected] (R. Hernández-Matehuala).





Background: Millepora alcicornis is a hydrocoral witha characteristic branching-growth pattern common in coralreefs of the Mexican Caribbean. Like other members of thisgenus, M. alcicornis is capable of inducing skin eruptionsand blisters with severe pain after contact. At present, verylittle is known about the systemic toxicity induced by thishydrocoral. Therefore in this study we investigated thesystemic toxic effects and the histopathological changesproduced by the aqueous extract of M. alcicornis in mice.We also assessed the toxic effects of this extract towardsthe nauplii of the brine shrimp Artemia salina and thedamages caused by the extract on this crustacean.

Methods: The acute toxic effects were analyzed in miceafter intravenously administration of the extract and thehistopathological changes were observed by light micros-copy. We perform cytotoxicity assay in Artemia salina andthe damages caused by the extract on the brine shrimpwere observed by Scanning Electron Microscopy (SEM) andTransmission Electron Microscopy (TEM).

Results: We found that the extract is lethal to mice(LD50 ¼ 17 mg protein/g) and caused kidney, liver, and lungdamage. In addition, this extract is toxic to A. salina (LD50 ¼70.71 mg protein/ml) and provoked several histologicalalterations in A. salina tissues.M. alcicornis aqueous extractcompletely lost its toxicological activity after incubation ina boiling water bath for 20 min.

Conclusions: These results indicate that theaqueous extract of M. alcicornis contains heat labile cytoly-sins that induce lethal effects inmice and in brine shrimps. Itis very likely that these toxins are also responsible for thedamages produced on mice and crustaceans tissues.

Financial support: Thisworkwas supported bygrant CB-2009-01 (project 133785) to Alejandra Rojas from the Con-sejo Nacional de Ciencia y Tecnología (CONACYT).

10.1016/j.toxicon.2012.04.123

H. Microbial Toxins

123. Effects of Cyanobacterial Bloom on Fish:Proteomics and Histological Investigation on theMedaka Oryzias Latipes

Marc Edery 1, Arul Marie 2, Benjamin Marie 1,Hélène Huet 3, Isabelle Trinchet 1, Lionel Dubost 2,Sahima Hamlaoui 1, Chakib Djediat 41UMR 7245 CNRS Molécules de communication et adaptation desmicroorganismes, équipe Cyanobactéries, Cyanotoxines et Environnement,Muséum National d'Histoire Naturelle, Paris, France2 Plateforme de spectrométrie de masse et de protéomique, Muséum Nationald'Histoire Naturelle, Paris, France3 Laboratoire d'Anatomie Pathologique, Ecole Nationale Vétérinaire d'Alfort,Maisons-Alfort, France4 Plateforme de microscopie électronique, Muséum National d'HistoireNaturelle, Paris, FranceE-mail address: [email protected] (M. Edery).

Background: Cyanobacterial toxic blooms oftenoccur in freshwater lakes and constitute a potentialhealth risk to human population, as well as to fishcommunity or other aquatic organisms. Microcystin-LR

(MC-LR, the most commonly detected cyanotoxin in thefreshwater environment) is a potent hepatotoxin, dereg-ulating kinase pathway via phosphatases 1 and 2A inhi-bition. Although toxicological effects have been clearlyrelated to the in vitro exposure of fish to purified micro-cystins, cyanotoxins are produced by the cyanobacteriatogether with numerous other potentially toxic molecules,and their global and specific implications on the health offish are still not clearly established and remain puzzling toassess.

Methods:Medaka fish (Oryzias latipes)was chosen as anin-vitro model for studying the effects of a cyanobacterialbloom on liver protein contents using a gel-free quantita-tive approach, iTRAQ, in addition to anatomic-pathologicalinvestigations. Fish were gavaged with cyanobacterialextracts (Planktothrix agardhii) from a natural bloom (Lakeof La Grande Paroisse, France) containing 2.5 mg eq. MC-LRper 5 ml. Two hours after exposure, fish were sacrificed andorgans were collected for analysis.

Results: Using proteomic approach, 304 proteins couldbe identified in treated fish livers, 147 presentinghigh confidence identification, among which 15 revealedstatistically significant variations in comparison withcontrols (gavaged with water only). Overall, these proteinregulations clearly testify to the occurrence of oxidativestress and lipid regulation effects in the liver of medakafish. Differently to pure mycrocystin-LR gavage experi-ments, a strong induction of Vitellogenin1 protein wasobserved for the first time with a cyanobacterial extract.This result was confirmed by ELISA quantification of vitel-logenin liver content.

Conclusions: These results suggest the occurrence ofan endocrine disruptor effect of estrogen-type of thePlanktothrix bloom extract, whether this reproductiveeffect can be attributed to microcystins need furtherinvestigation.

Keywords: medaka, cyanobacteria, proteomics10.1016/j.toxicon.2012.04.124

124. Biogeography of Microcystins

Cristiana Moreira 1,2, Vitor Vasconcelos 1,2,Agostinho Antunes 1,2

1Department of Biology, Faculty of Sciences, Porto University, Portugal2Marine and Environmental Research Center – CIIMAR/CIMAR, PortoUniversity, PortugalE-mail address: [email protected] (V. Vasconcelos).

Review: Microcystins are toxins produced by cyano-bacteria that can be lethal to humans as well as wildanimals and livestock. Microcystins are cyclic heptapep-tides and are found in all the main continents, beingfirstly recorded in the species Microcystis aeruginosa.Biogeography of toxins is a may help determine anybiogeographic pattern that can explain the distribution ofa certain toxin. In virtue of the lack of knowledge underthis topic we conducted a study to assess if there is anybiogeographical trend for this cyanotoxin on a worldwidescale. Three genes involved in the biosynthesis ofmicrocystins (mcyA, mcyD, and mcyG) were retrieved




from the worldwide databases and were geographicallyrelated using phylogenetic tools. Results for all threegenetic markers indicate the occurrence of a southernhemisphere cluster (South Africa and Australia), Asia hada distinct population from other continental groups andthese appear to have no stringency. These data bring tolight the importance of evaluating the biogeography ofnatural occurring toxins since their distribution can beclearly influenced by geography. Events such as specia-tion and dispersal are phenomena that can explain theirpresent occurrence.

Financial support: This research was funded bythe PTDC/AAC-AMB/104983/2008 (FCOMP-01-0124-FEDER-008610) and PTDC/AAC-CLI/116122/2009 (FCOMP-01-0124-FEDER-014029) projects from Fundação paraa Ciência e Tecnologia (FCT), and by the PhD fellowshipto Cristiana Moreira (Ref. SFRH/BD/47164/2008) fromFCT.

Keywords: phylogeny, biogeography, M. aeruginosa.10.1016/j.toxicon.2012.04.125

125. Cadherin Binding Is Not a Limiting Step forBacillus thuringiensis subs. israelensis Cry4BaToxicity to Aedes aegypti Larvae

Claudia Rodríguez-Almazán 1, Esmeralda Z. Reyes 1,Isabel Gómez 1, Amy M. Evans 2,Supaporn Likitvivatanavong 2, Alejandra Bravo 2,Sarjeet S. Gill 2, Mario Soberón 1

1 Insituto de Biotecnología, Departamento de Microbiología Molecular,Universidad Nacional Autónoma de México, Cuernavaca, Morelos, México2Department of Cell Biology and Neuroscience, University of California,Riverside, California, USAE-mail address: [email protected] (C. Rodríguez-Almazán).

Background: Bacillus thuringiensis ssp. israelensis (Bti)produces insecticidal proteins known as Cry toxins(Cry4Aa, Cry4Ba and Cry11Aa). These toxins are highlyspecific to their target insect, and are innocuous to verte-brates and plants. Cry toxins are pore-forming toxins thatinteract with specific receptor located on the host cellsurface. Cry proteins are ingested by susceptible larvaedissolve in the alkaline environment of the gut, therebyreleasing soluble protoxins, these are cleaved at specificsites by midgut proteases yielding active protease-resistantfragments. These fragments bind to specific proteinreceptors on midgut epithelial cells (cadherin receptor andGPI-anchor receptor), leading to membrane insertion andpore formation producing death of the insect. This mech-anism is accompanied by toxin oligomerization beforemembrane insertion. Previous work showed that Cry11Aabinds to cadherin receptor fragment (CR7-11) with highaffinity. Binding to cadherin has been proposed to facilitateCry toxin oligomer formation.

Methods: In the present work we analyzed binding ofCry4Ba and Cry11Aa to cadherin receptor (CR7-11). Wedetermined the affinity by ELISA binding assay and bysurface plasmon resonance (SPR).

Results: Cry4Ba binds to CR7-11 in a saturable way withnine fold lower binding affinity (Kd of 134.9 nM by ELISA

and Kd of 154 nM by SPR) than that of Cry11Aa (Kd of 14.8nM by ELISA and Kd of 17 nM by SPR). In the oligomerformation assays, only in presence CR7-11, fragmentinduced oligomer formation in Cry11Aa in contrast toCry4Ba that oligomers were formed evenwhen activated inthe absence of CR7-11. Pore formation of Cry4Ba oligomerswas determined in black lipid bilayers showing theformation of ion channels. We analyzed the toxicity toAedes aegypti of both toxins in the presence of CR7-11.Cry11Aa toxicity was enhanced 2.7 fold in the presence often fold excess of CR7-11. In contrast, there was no changein Cry4Ba toxicity. Finally, silencing the cadherin gene bydsRNA showed that silenced larvae were more tolerant toCry11Aa in contrast to Cry4Ba, that showed similar toxiclevels to control larvae.

Conclusions: Although cadherin binding has beenshown to be an important binding step of different Crytoxin, the data presented here shows that it is not a limitingstep in the toxicity of Cry4Ba to A. aegypti.

Keywords: Cry toxins, receptor binding, Bacillus thuringiensis.10.1016/j.toxicon.2012.04.126

126. Magnetic Resonance Imaging and SpectroscopyStudy of Microcystin LR Hepatotoxicity

Jerneja Strupi �Suput 1, Igor Ser�sa 2, Du�san �Suput 11 Institute for Pathophysiology, Faculty of Medicine. University of Ljubljana,Slovenia2 Institute Jozef Stefan, Jamova 13. Ljubljana, SloveniaE-mail address: [email protected] (D. �Suput).

Background: Acute lethal intoxicationwith microcystinLR (MC-LR) results in hepatocyte damage and intra-hepatichemorrhage. Hepatotoxic effects of microcystins have beenstudied in detail, and inhibition of protein phosphatasesseems to be responsible for the majority of the observedeffects. However, very little is known about the metabolicchanges in hepatocytes of an experimental animal exposedto those toxins. Apoptosis of hepatocytes and blood stasismay decrease the level of ATP and creatine phosphate inhepatocytes of experimental animals. The aim of thepresent study was tomeasure the changes in liver structureand relative concentrations of energy rich metabolites inthe liver of experimental animals noninvasively usingmagnetic resonance imaging (MRI) and P31magneticresonance spectroscopy (MRS).

Methods: Adult male mice strain C57BL/6J (The Jack-son Laboratory, USA) age 11-15 weeks, weighting from19-27 g were used for P31 magnetic resonance spectros-copy, and Sprague-Dawley rats weighting 190 to 240 gwere used for MR imaging. Before the experiment theanimals were sedated and anesthetized by xylazine(Chanasine, Chanelle Pharmaceuticals, Ireland) and ket-amine (Bioketan, Vetoquinol Biowet, Poland). Measure-ments of liver morphology changes by means of MRI andmeasurements of phosphorous compounds by P31 MRSwere performed on a small bore 2,35 T Bruker tomographbefore and after the animals had received a LD50 of MC-LRintraperitoneally.




Results: Exposure of experimental animals to micro-cystins caused a dose and time - dependent increase ofinorganic phosphate with corresponding decrease of crea-tine phosphate and ATP peaks. In animals thathave survived the treatment the changes were only minoror even absent. MR imaging showed an increased signalintensity from the liver of animals exposed to MC-LR. Anincrease in the liver size was most evident in animals thatdied due to MC-LR intoxication. Post mortem investigationshowed typical pathohistological changes.

Discussion: Previous data have shown that MRI candetect changes in liver morphology due to higher protoncontent in the affected portions of the liver. Our datashow strong correlation between pathohistological find-ings and MRI, and P31 MRS showed a decrease in theconcentration of energy-rich such as ATP and creatinephosphate with concomitant increase of inorganicphosphate.

Conclusions: This study confirms that MC-LR increasesthe dephosphorylation of energy rich substances such asATP and creatine phosphate and impairs the adequategeneration of ATP. This is in agreement with the previousfindings that microcystins cause mitochondrial swellingand induce apoptosis, which are energy-demandingprocesses.

Keywords:microcystin, liver, magnetic resonance, imaging, spectroscopy, P3110.1016/j.toxicon.2012.04.127

127. Urease of Helicobacter pylori: Roles inInflammation and Platelet Activation

Augusto F. Uberti 1, Deiber Olivera-Severo 1,Adriele Scopel-Guerra 1, Christina Barja-Fidalgo 2,Celia R. Carlini 1,31Graduate Program in Cellular and Molecular Biology – Center ofBiotechnology, Universidade Federal do Rio Grande do Sul, Porto Alegre,RS, Brazil2Dept. Pharmacology, Universidade Estadual do Rio de Janeiro, Rio deJaneiro, RJ, Brazil3Dept. Biophysics & Center of Biotechnology, Universidade Federal do RioGrande do Sul, Porto Alegre, RS, BrazilE-mail address: [email protected] (C.R. Carlini).

Background: Ureases (EC 3.5.1.5), nickel-dependentenzymes that hydrolyze urea into ammonia and CO2, aresynthesized by plants, fungi and bacteria. The ureaseproduced by Helicobacter pylori (HPU), an etiological agentof gastric ulcers and cancer, is considered a virulence factorsince its ureolytic activity enables the bacterium to survivein the acidic medium of the stomach. Gastric colonizationby H. pylori is usually accompanied by an intense infiltra-tion of polymorphonuclear leukocytes, macrophages andlymphocytes. The degree of mucosal damage correlateswith an intense neutrophil infiltration. It is well known thatplatelets participate in the inflammatory response bymodulating the activity of other inflammatory cells andischemic lesions due to vascular insufficiency may lead toulcers within the gastric mucosa. Previous data of ourgroup showed that HPU: 1) induces platelet aggregationindependent of its ureolytic activity, requiring ADP secre-tion through the lipoxygenase pathway; 2) induces paw

edema in a dose- and time-dependent manner, with anintense neutrophil infiltration.

Methods: In this work, we used a recombinant ureaseproduced in Escherichia coli to evaluate biological effectsindependent of its enzyme activity.

Results: In platelets, our results indicate that HPUinteracts with themembrane glycoprotein VI, a well knownreceptor for collagen, which triggers a signaling cascaderequiring metabolites of the platelet 12-lipoxygenasepathway. In human neutrophils, 100 nM rHPU inducedextracellular production of ROS, and inhibited theirapoptosis (40.5% compared to control), altering the levels ofBcl-XL and Bad proteins. HPU-induced neutrophil chemo-taxis was 88% of that observed by fMLP, a strong chemo-attractant used as positive control. These effects of rHPUpersisted in the absence of enzyme activity. rHPU-inducedpaw edema, neutrophil chemotaxis and apoptosis inhibi-tion reverted in the presence of the lipoxygenase inhibitorsesculetin or AA861. Neutrophils exposed to rHPU hadincreased content of lipoxygenase(s) and no alteration ofcyclooxygenase(s) level(s).

Conclusion: Our data indicate that HPU, besidesallowing the bacterial survival in the stomach, could playan important role in the pathogenesis of the gastrointes-tinal inflammatory disease caused by H. pylori.

Financial support: CAPES, CNPq, FAPERJ and FAPERGS.

Keywords: urease, Helicobacter pylori, inflammation10.1016/j.toxicon.2012.04.128

128. Human iPS Neuronal Platform for BotulinumNeurotoxins

Eric A. Johnson, Sabine Pellett, Regina Whitemarsh,William H. TeppDepartment of Bacteriology, University of Wisconsin, Madison, WI, USAE-mail address: [email protected] (E.A. Johnson).

Background: Human induced pluripotent stem (hiPS)cells hold great promise for providing various differentiatedcell models for in vivo toxigenicity testing. For Clostridiumbotulinum neurotoxin (BoNT) detection and mechanisticstudies several cellmodels currently exist, but none examinetoxin function with species-specific relevance while exhib-iting high sensitivity. The most sensitive cell models to dateare mouse or rat primary cells and neurons derived frommouse embryonic stem cells, both of which require signifi-cant technical expertise for cell preparation.

Methods: This study describes for the first time the use ofhuman iPS derived neurons for BoNT detection. The neuronsare already differentiated and cryopreserved and consist ofa 98% pure pan-neuronal population of GABAergic, dopa-minergic, and glutamatergic neurons (Cellular Dynamics Inc,Madison, WI). Western blot and qPCR data show that thecells express all the necessary receptors and substrates forBoNT intoxication by all BoNT serotypes.

Results: BoNT/A intoxication studies demonstrate thatthe iPS-derived neurons detect biologically active BoNT/Areproducibly, quantitatively, and with high sensitivity(EC50 w 0.3 Units). Specificity was confirmed by antibody




protection studies, and quantitative detection of BoNTserotypes B, C, and E as well as BoNT/A complex aredemonstrated. Direct comparison to BoNT detection usingprimary rat spinal cord cells showed equal or increasedsensitivity and a steeper dose-response curve, reaching100% SNARE protein target cleavage with significantly lessBoNT.

Discussion: These data suggest that neuronsderived from human iPS cells provide an ideal andhighly sensitive platform for BoNT potency determina-tion, neutralizing antibody detection, as well as formechanistic studies of other neurotoxins of diversecomposition.

Keywords: neurotoxins, pluripotent stem cells, botulinum neurotoxin10.1016/j.toxicon.2012.04.129

129. Retrograde Trafficking at the Presynaptic NerveTerminal Using Bacterial Toxins

Sally Martin, Callista B. Harper, Frederic A. MeunierQueensland Brain Institute, The University of Queensland, St Lucia,Brisbane, QLD, AustraliaE-mail address: [email protected] (S. Martin).

Background: A comprehensive understanding ofthe regulation of presynaptic sorting and retrogradetransport of membrane vesicles is vital for understandingneuronal development, plasticity and survival. Theappropriate sorting of receptors and ligands at the nerveterminal can occur at the cell surface or followingendocytosis, through incorporation into specificmembrane domains, mechanisms that are also used bytoxins.

Methods: To analyse presynaptic membrane sorting wehave used two probes based on bacterial toxins, whichtarget the local recycling and retrograde pathways witha high degree of specificity. The retrograde pathway wastargeted using the atoxic B-subunit of cholera toxin (CTB),and the local pathway using the atoxic heavy chain ofbotulinum A (BoNT/A-Hc). Endocytosis and membranesorting was analysed in primary hippocampal neurons byboth fluorescence and live cell microscopy, in addition toelectron microscopy.

Results: Both BoNT/A-Hc and CTB enter neuronspredominantly at the presynaptic nerve terminal. Entryof BoNT/A-Hc is activity-dependent. Entry of CTB isrestricted to a subset of nerve terminals in restingneurons, but following membrane depolarisation CTBenters all nerve terminals. CTB and BoNT/A-Hc aresegregated at the nerve terminal, both at the level of thecell surface and in intracellular vesicles. CTB entersretrograde carriers that are contain the neurotrophinreceptor, TrkB, and undergoes retrograde transport to theGolgi complex in the cell body. BoNT/A-Hc predomi-nantly enters synaptic vesicles and early endosomes.BoNT/A-Hc endocytosis is dependent on dynamin,a GTPase involved in both clathrin-mediated and cla-thrin-independent endocytosis. Inhibiting the activity ofdynamin prevents BoNT/A-Hc endocytosis and slows the

development of botulism in mice (Harper et al. 2011 JBC286:35966).

Discussion: Understanding the molecular mecha-nisms that underpin the sorting and directed transport ofinternalized proteins at the presynaptic nerve terminal isimportant not only for understanding intoxication, butalso for understanding sorting and targeting of vitalsurvival factors. By using atoxic fragments of toxins thathijack neuronal pathways we are beginning to dissect outthe molecular machinery that underlies these processes.We have found that the sorting of proteins destined fordifferent destinations in the cell begins to occur veryearly in their trafficking, potentially as early as incorpo-ration into specific domains at the plasma membrane.Furthermore, we have demonstrated that specificallyinhibiting molecular components of the endocyticmachinery is a viable approach to identifying potentialtargets for therapeutic intervention.

Conclusion: Early regulation of endocytic carriersunderlies the fidelity of neuronal presynaptic membranesorting.

Keywords: trafficking, bacterial toxins, neurons10.1016/j.toxicon.2012.04.130

130. Water Bacterial Activities Involved in ImmuneDepression of Haemodialysis Patients

Meshref A. Al-Ruwaili, Samy A. SelimMicrobiology Laboratory, Department of Medical Laboratory Sciences,Collage of Applied Medical Sciences, Al-Jouf University, Sakaka, Saudi ArabiaE-mail address: [email protected] (M.A. Al-Ruwaili).

Background: The present work surveyed haemodialysis(HD) units, in order to assess the bacteriological and tox-inological qualities of dialysis water and dialysate; anddetermine to experimentally to how extent the bacterialactivities are involved in immune depression for thepatients.

Methods: Quantitative methods were used toenumerate the total count of viable heterotrophic bacteria;total coliforms; Pseudomonas spp.; Aeromonas spp. (cfu/ml); and endotoxin concentration (EU/ml) in dialysis waterand dialysate of HD centers.

Results: The most commonly isolated Gram-negativebacteria from treated water were Aeromonas spp., 22.2.Endotoxin concentrations ranged between 3 to 113 (EU/ml)for water and dialysate respectively. The haematologicalanalysis had non significant except haemoglobin level hada significant values (P¼ 0.02). The clinical data showedcomparable difference than control patients. Missing, out ofwork, bypassed and even erroneously arranged treatmentfacilities are some of the reasons responsible for thedetected high levels of bacterial and endotoxin in finalproduct water.

Conclusions: The presence and growth of bacteria inwater and dialysate should be controlled and the endotoxintesting must be a part of the regular quality control regime,to minimize the risk of adverse reactions in HD patients.



Fig. 1. Percentage of some bacterial toxins, specific antibodies, and pyro-genic reactions in haemodialysis patients.

Table (1): Mean values of total heterotrophic bacteria, total coliforms,Pseudomonas spp. and Aeromonas (cfu/100ml), endotoxins (EU/ml) inwater and dialysate of haemodialysis centers.

Tap Water Treated Water Dialysate

Total heterotrophic bacteria 197.8 234.2 101Total coliforms 71.5 21. 5 2Pseudomonas spp. 37.3 9.2 14.4


Keywords: bacteriological, toxinological, immune depression, haemodial-ysis patients10.1016/j.toxicon.2012.04.131

Aeromonas spp. 56.2 22.2 9.7Endotoxins 3 17 13

Fig. 1. DNS sequences of 348-bp region of cytochrome b gene from sus-pected fish sample and accession No. HQ630754.1 of Tetrapturus audax(striped marlin).

131. Unexpected Hazard Due to Fuminaisin ToxinContaminating Herbal Teas in Saudi Arabia

Fardos Bokhari 1, Magda M. Aly 2

1Biology Department, Faculty of Science, King Abd El-Aziz University,P. O. Box 12161, Jeddah, Kingdom Saudi Arabia2Botany Department, Faculty of Science, Kafr El-Sheikh University, EgyptE-mail address: [email protected] (F. Bokhari).

Background: Fumonisins are phytotoxic mycotoxinsthat are synthesized by various species of the genusFusarium and are hazardous for human and animal health.The purpose of this study was to investigate fumonisin B1(FB1) in herbal tea consumed especially by Saudipopulation.

Methods: FB1 was detected using immunoabsorpantcolumn chromatography with fluorescence detection. 40commercially available samples were collected andanalyzed for FB1.

Results: The detectable amount for FB1 was rangedfrom 0- 266 ppm. All the herbal tea samples were eval-uated for the fungal contamination and the presence ofmycotoxigenic fungi. Results indicated that predominantmycoflora was distributed in 13 genera and 20 species.From these, the genera Aspergillus, Penicillium and Fusa-rium that considered extremely important from themycotoxicological standpoint were the most abundantfungi.

Conclusions: The presence of toxigenic mouldsrepresents a potential risk of other mycotoxin contam-ination and considering the worldwide increased use of

herbal products as alternative medicines, it is necessarysetting standards for toxigenic moulds in crude herbaltea in order to reduce the risks for consumers' health.

Keywords: fumonisins, herbal tea, toxigenic moulds, mycotoxins10.1016/j.toxicon.2012.04.132

132. Determination of Histamine and Histamine-forming Bacteria in Striped Marlin Fillets (Tetrapturusaudax) Implicated in a Food-borne Poisoning

Yi-Chen Lee 1, Tzou-Chi Huang 1, Chung-Saint Lin 2,Chia-Min Lin 3, Yung-Hsiang Tsai 31National Pingtung University of Science and Technology, Pingtung, Taiwan2 Yuanpei University, Hsin-Chu, Taiwan3National Kaohsiung Marine University, Kaohsiung, TaiwanE-mail address: [email protected] (Y.-H. Tsai).

Background: An incident of food borne poisoningcausing illness in 67 victims due to ingestion of fried fishfillets occurred in June, 2011, in Kaohsiung city, southernTaiwan.

Methods: Epidemiological, bacterial and chemicalinvestigations were performed.

Results: Of the five suspected fish fillets, two samplescontained 62.0-mg/100 g (fried sample) and 89.6-mg/100g (raw sample) of histamine, which is greater than



Fig. 2. Histamine residual percentage of marlin fillet implicated in foodpoisoning at 89.6 mg/100 g histamine by frying for 0, 1, 2, 3, 4, 5 min. Barsrepresent mean þ/� SD of three replications. Bars with the different lettersare significantly different (p<0.05).


50 mg/100 g of the potential hazard level in most illnesscases. Given the allergy-like symptoms of the victims andthe high histamine content in the suspected fish samples,

Table 1Values of the pH, water content, salt content, aerobic plate count (APC), total volatimplicated in food poisoning.

Sample type and no. pH Water content (%) Salt content (%) T

Fried filletsF-1 6.04 62.2 0.81 2F-2 6.09 65.6 0.88 1F-3 6.19 59.3 0.88 1Raw filletsR-1 6.24 85.5 0.66 2R-2 5.98 87.2 0.61 1

Table 2The levels of biogenic amines in the marlin fillets implicated in food poisoning.

Sample type and no. Levels of biogenic amine (mg/100 g)

Put a Cad Try Phe

Fried filletsF-1 2.7 0.2 ND b NDF-2 0.4 0.5 ND NDF-3 1.1 0.2 ND NDRaw filletsR-1 2.7 0.5 ND NDR-2 3.0 2.7 ND ND

a Put, putrescine; Cad, cadaverine; Try, tryptamine; Phe, 2-phenylethylamine;agmatine.

b ND, not detected (amine level less than 0.05 mg/100g).

Table 3Identification of histamine-forming bacteria isolated from the uncooked marlin fi

from NCBI database analysis, and their production of histamine and other bioge

Strain Organism identified Percentage identity (%) Source

R-1-1 Raoultella ornithinolytica 100 R-1R-1-2 Raoultella ornithinolytica 100 R-1R-1-3 Enterobacter aerogenes 100 R-1R-1-4 Enterobacter aerogenes 100 R-1R-2-1 Morganella morganii 100 R-2

a His, histamine; Put, putrescine; Try, tryptamine; Spd, spermidine.b ND: Not detected (amine level less than 0.05 ppm).

this food-borne poisoning was strongly suspected to becaused by histamine intoxication. Five histamine-producing bacterial strains capable of producing 59 to 562ppm of histamine in trypticase soy broth (TSB) supple-mented with 1.0 % L-histidine (TSBH) were identified asEnterobacter aerogenes (two strains), Raoultella ornithino-lytica (two strains) and Morganella morganii (one strain).The fish species of suspected samples were identified asstriped marlin (Tetrapturus audax) by using PCR directsequence analysis. In addition, the degradation loss ofhistamine in suspected raw fillets was 28% after 170 �Cfrying for 5 min.

Conclusions: An outbreak of Scombroid foodpoisoning was confirmed by clinical symptomatology,epidemiologic investigation, and chemical and bacterio-logical analyses.

Keywords: histamine; striped marlin; histamine-forming bacteria; scom-broid poisoning10.1016/j.toxicon.2012.04.133

ile basic nitrogen (TVBN), total coliform (TC), and E. coli in the marlin fillets

VBN (mg/100g) APC (log CFU/g) TC (MPN/g) E. coli (MPN/g)

1.1 2.70 <3 <37.2 5.74 10 <37.2 3.04 <3 <3

1.8 8.02 340 <33.9 7.79 130 <3

Spd Spm His Tyr Agm

2.1 ND 17.8 1.2 NDND ND 62.0 3.0 ND0.4 ND 3.8 1.2 ND

1.1 ND 89.6 1.2 ND0.5 ND 0.8 0.5 ND

Spd, spermidine; Spm, spermine; His, histamine; Tyr, tyramine; and Agm,

llets implicated in food poisoning by 16S rDNA, based on the output resultsnic amines (ppm) in culture broth.

GenBank accession number His* Put Try Spd

HQ242731.1 562 2.6 0.2 0.1HQ242730.1 129 4.6 2.4 0.3JF430156.1 287 2.5 2.0 1.1AB244467.1 246 24.8 NDb 0.3JF947361.1 59 5.0 1.3 1.7


133. Root Toxicity of the Mycotoxin Botryodiplodin inSoybean Seedlings

W. Thomas Shier 1, Justin Nelson 1, Hamed K. Abbas 2,Richard E. Baird 3

1College of Pharmacy, University of Minnesota, Minneapolis, MN, USA2US Dept. of Agriculture, Agricultural Research Service, Stoneville, MS, USA3Dept. of Entomology and Plant Pathology, Mississippi State University,Mississippi State, MS, USAE-mail address: [email protected] (W.T. Shier).

Background: The fungus Macrophomina phaseolinacauses disease in more than 500 plant species. Insoybean it causes charcoal rot disease, which is the majorcause of soybean yield losses in Mississippi and a signifi-cant problem around the world, particularly under dryconditions. M. phaseolina infects plants from the reservoirof inoculum in soil by entering plants through theroots. Once inside, it extends mycelium through thevascular system to other parts of the plant. As previouslyreported, M. phaseolina isolated from infected plants andsoil in southeastern USA produces the mycotoxin(-)-botryodiplodin, but not phaseolinone. (-)-Botryodi-plodin is believed to be a virulence factor that M. pha-seolina uses to penetrate roots during the initial infectionprocess. The fungus secretes mycotoxin, which killsnearby plant tissue, allowing the fungus to easily enterthe plant through the necrotic tissue produced by thetoxin.

Methods: The effects of (�)-botryodiplodin on soybeanroot were investigated in soybean seedlings germinated insoil and transplanted at the cotyledon stage (VC, 4-7 cmgrowth) to hydroponic growth in individual glass tubes in10% Villagarcia medium.

Results and Discussion: During 4 days growth atroom temperature in continuous light, submergenceinduced abundant lateral root growth. The seedlingswere transplanted to fresh medium containing a rangeof concentrations (0-80 mg/ml) of (�)-botryodiplodinprepared by chemical synthesis, and cultured an addi-tional 4 days. Lateral root growth was inhibited at �4 mg/ml (�)-botryodiplodin. (�)-Botryodiplodin at 80 mg/mlcaused severely stunted roots that were stained pink, thecolor of the pigment produced when botryodiplodinreacts with protein. Inhibition of root growth was quan-tified by excising roots, drying lateral and tap rootsseparately and weighing. (�)-Botryodiplodin inhibitedtap root growth at IC50 ¼ 23.5 mg/ml and lateral rootgrowth at IC50 ¼ 4.2 mg/ml. There were no apparent toxiceffects to aerial parts of the plants, except secondary toroot loss.

Conclusions: The results were consistent withbotryodiplodin selectively killing actively growingmeristematic tissue at root tips. Production of necrotictissue at root tips would be expected to provide thefungus with ready access to the plant vascular system.Supported in part by the Mississippi Soybean PromotionBoard.

Keywords: mycotoxin, botryodiplodin, soybean10.1016/j.toxicon.2012.04.134

134. Cytotoxic and Proteolytic Molecules of the HumanParasite Dientamoeba fragilis, Identified by RNA seq,Provide Support for its Pathogenic Capacity

Joel Barratt 1, Damien Stark 1,2, John Ellis 1

1 The University of Technology, Sydney (UTS), Sydney, Australia2 St Vincent's Hospital, Sydney, AustraliaE-mail address: [email protected] (J. Barratt).

Background: Dientamoeba fragilis is a single-celledgastrointestinal parasite of humans with a worldwidedistribution. Since its discovery Dientamoeba has beenlargely neglected in clinical circles due to doubtssurrounding its pathogenicity. Knowledge regarding themolecular biology of Dientamoeba is scarce and only 34 DNAand cDNA sequences (collectively) are available for Dien-tamoeba in Genbank (at the time of writing). Despitenumerous reports of clinically apparent dientamoebiasis, noresearch has been conducted to identify the mechanisms ofpathogenicity employed by this organism. It is well estab-lished that various pathogenic helminths (worms) and othersingle-celled human parasites excrete/secrete a range ofcytotoxic and proteolytic molecules which induce hosttissue destruction and host cell lysis. These moleculesrepresent important factors associated with pathogenicity. Itwas postulated that Dientamoeba also excretes similarcytotoxic and proteolytic molecules and that these mole-cules are involved in the pathogenisis of clinically apparentdientamoebiasis. This hypothesis was explored using highthroughput sequencing technologies to determine whetherDientamoeba expresses similar cytotoxic factors in vitro.

Methods: Dientamoeba parasites were isolated intoxenic culture systems from the stools of patients presentingat St Vincent's Hospital, Sydney (Australia), with gastroin-testinal complaints. Total RNA was extracted from culturesusing standard Tri-reagents. Enrichment of eukaryoticmRNA's from total RNA extracts was achieved using oligo-(dT) chromatography. These mRNA's were sequenced usingRoche GS FLX sequencing technology. Sequence reads wereassembled using Newbler (version 2.6). The identity ofresulting contigs/isotigs was predicted using the Blast2GOprogram. Homologs to specific cytotoxic/cytolytic mole-cules were also identified using a standalone version of theNCBI tblastn algorithm.

Results: Four cultures of Dientamoebawere obtained andtwo were selected for sequencing. Assembly of sequencingreads yielded approximately 6000 contigs/isotigs (dependingon the assembly settings/paramaters). Homology searchesusing Blast2GO and tblastn identified a repertoire of Dien-tamoeba molecules which are potentially involved in patho-genic processes. These molecules bear strong homology tofactors produced by other pathogenic parasites, which areinvolved in host tissue destruction and host cell lysis.

Conclusions: These preliminary results indicate thatDientamoeba produces molecules which are potentiallyinvolved in pathogenic processes, though further charac-terisation of these molecules is required. This will involvepurification of these proteins followed by in vitro cytotox-icity assays involving mammalian cell lines to determinetheir true cytotoxic/cytolytic activity. This is the first reportdescribing potentially cytotoxic and proteolytic molecules




in Dientamoeba. Importantly, this data provides support forthe pathogenic capacity of Dientamoeba fragilis.

Keywords: Dientamoeba fragilis, cytotoxin, cytotoxic, cytolytic, proteolytic,gastrointestinal, parasite10.1016/j.toxicon.2012.04.135

135. Evaluation of Cardiotoxic Effects of Microcystin–LRin Mouse Isolated Hearts

F. Siqueira-Lece 1, H.D. Ricardo 1, M.A. Tomaz 1,M.M. Machado 1, J.M.1,2, S.M. Tavares 1, M.A. Strauch 1,S.M. Azevedo 2, R.M. Soares 2, P.A. Melo 1

1 Lab. Farmacologia das Toxinas, ICB, CCS, UFRJ, Rio de Janeiro, RJ Brazil2 Laboratory of Ecophysiology and Toxicology of Cyanobacteria, HealthSciences Center, Federal University of Rio de Janeiro, BrazilE-mail address: [email protected] (P.A. Melo).

Background: Microcystin-LR (MC-LR) is related toenvenoming of animals and humans following blooms ofcyanobacteria and the release of large quantities of thetoxin in lakes and rivers used as water supplies. There areno previous studies showing the cardiac effects of MC-LRunder oxidative stress conditions, such as ischemia andreperfusion (I/R). Themain goal of this studywas to analyzethe effect of MC-LR on isolated mouse heart cardiacfunction.

Methods: We assessed the effects of MC-LR in mouseisolated hearts perfused with an appropriated nutritionalsolution by using the modified Langendorff preparation.We analyzed the tension developed, the electrocardio-graphic records (EKG), the damaged area and the CreatineKinase (CK) activity in the perfusate. The preparation wasperfused under control conditions and after 15 minutes ofstabilization, we added different concentrations MC-LR(0.1-0.3 mg/mL) to the bathing media after 10 min of I/R. Atthe end of the experiments, hearts were gently slicedand exposed to 1% triphenyl tetrazolium chloride (TTC) toassess damaged areas (Am Heart J, 593: 101, 1981). Elec-trical and contractile properties were analyzed by theWINDAQ program.

Results: The heart exposure at MC-LR 0.1mg/mL did notshow any changes. At concentrations 0.3mg/mL MC-LRdecreased more than 60% on the cardiac tension and on theQRS waves, after 70 minutes compared to control heartsexposed to nutritional solution (n¼4). The analysis withTTC test did not show image evidences of damage, althoughthe CK released in the perfusate increased more 100%. TheI/R protocols did not change the control records or the TTCimage or CK analysis. However, the hearts exposed to 0.1mg/mL after I/R, showed a decrease of 50% of the cardiactension without changes on the EKG wave sizes or CKreleased.

Discussion: Our experiments showed that MC-LR alonehas cardiotoxic effect above 0.3 mg/mL and its cardiotoxiceffects is increased by I/R conditions which stress thecardiac tissue.

Conclusions: Our results have shown for the firsttime the direct damage and effects on cardiac functionby MC-LR and this effect is increased by oxidative stressconditions.

Financial support: CAPES, CNPq, PRONEX, MCT/INCT -INPeTam and FAPERJ.

Keywords: Cyanobacteria, Microcystin LR., mouse isolated heart, ischemiaand reperfusion10.1016/j.toxicon.2012.04.136

I. Miscellaneous

136. The Allosteric Binding Site for r-TIA on theExtracellular Surface of the a1B- Adrenoceptor

Lotten Ragnarsson 1, Ching-I. Anderson Wang 1,Åsa Andersson 1, Dewi Fajarningsih 1, Thea Monks 1,Andreas Brust 1,2, K. Johan Rosengren 3, Richard J. Lewis 1

1University of Queensland, Institute for Molecular Bioscience, Brisbane,Queensland 4072, Australia2Xenome Ltd., Indooroopilly, Queensland, Australia3University of Queensland, School of Biomedical Sciences, Brisbane,Queensland, AustraliaE-mail address: [email protected] (R.J. Lewis).

Background: The G protein-coupled receptor (GPCR)superfamily comprise over 1000 membrane receptorsthat functionally couple extracellular stimuli to intracel-lular effectors to control many vital processes. Despitethe potential of extracellular surface (ECS) residues inClass A GPCRs to interact with subtype specific allostericmodulators, an ECS pharmacophore has not beenidentified.

Methods: Using the turkey beta1-adrenergic receptorstructure, we built a newmodel of the alpha1B-adrenoceptor(a1B-AR) to help identify the allosteric modulatory site ofrho-conopeptide TIA, an inverse agonist at this receptor.

Results: Combining mutational, radioligand bindingand IP-one signaling studies together with moleculardocking simulations using a refined NMR structure of r-TIA,we identified eight residues on the extracellular surface(ECS) of the a1B-AR that influenced rho-TIA binding.Double mutant cycle analysis and docking confirmed thatrho-TIA bindingwas dominated by a salt bridge and cation-p and T-stacking-p interactions.

Conclusions: These interactions reveal that ligandsbinding to extracellular loop 2 are sufficient to allostericallyinhibit agonist signaling at a GPCR. The ligand-accessibleECS residues identified provide the first view of an ECSpharmacophore of a GPCR and the first set of structuralconstraints for the design of novel allosteric antagonistsacting at the ECS of adrenergic receptors.

Keywords: conotoxin, adrenergic receptors, toxin binding determinants10.1016/j.toxicon.2012.04.137

137. Neuropathies of Spinal Cord Development of RatPups Maternally Fed on Fried Potato Chips

Gadallah A. Abdelalim 1, El-Sayyad I. Hassan 2,El-Shershaby M. Effat 2, Abdelatif M. Ibrahim 2

1 Preparatory Year Deanship, Jazan University, Jazan, KSA2 Faculty of Science, Dept. of Zoology, Mansoura University, Mansoura, EgyptE-mail address: [email protected] (G.A. Abdelalim).





Aims/Objectives: Recently, elevated levels of acryl-amide in variety of foodstuffs including fried potatoes chipswere reported. The study aimed to illustrate the neuropa-thies of and demyelination of spinal cord of pups mater-nally fed on diet containing fried potatoes chips.

Study design: Experimental study.Methodology: Eighty fertile virgin females and fertile

males of albinoWistar rats (Rattus norvegius). Females weremade pregnant after mating and zero date of gestation wasdetermined. They were arranged into three groups; control,acrylamide-treatment (15mg./kg B.wt) and diet containing50%fried potatoes chips. Pregnant were treated withacrylamide or fed on fried potatoes chips every other dayuntill 3 week post-partum. The cervical cord was separatedat 2 & 3week-old rat. Fresh sampleswere subjected for SDS-PAGE analysis; meanwhile other specimens were processedfor light and transmission electron microscopy (TEM).

Results: The present findings revealed altered proteinexpression in both experimental groups. Light and electronmicroscopic observations exhibited neuropathologicalalterations especially of ependymal canal and neuronalcells. Demyelination of axons was observed.

Conclusion: Maternal supplementation of fried potatoeschips during gestation and suckling period led to consumelarge amount of acrylamide generated during cocking aswellas its metabolite glycidamide. Both components find theirway across transplacental during gestation and breast milkduring lactation period interfering with spinal cord differ-entiation and cause neurotoxicity and demyelination.

Keywords: neuropathies, offspring, fried potatoes chips10.1016/j.toxicon.2012.04.138

Fig. 1. Effects of oral administration of ACE inhibitors and AT1R blocker for15 successive days before induction of hepatocarcinogenesis and throughoutthe experimental period on hepatic tumor markers AFP and CEA. Both tumormarkers were measured by ELISA kits. The results were expressed as ng/mL.Each column represents the mean of 10 rats with a vertical bar showing theSEM. *Significant difference from control group (p<0.05). #Significantdifference from DENA group (p<0.05).

138. Nickel hepatotoxicity in rats and trials forprotection using antioxidants

Salah S. El-Ballal 1, Ashraf M. Morgan 2, Nemin B. Ebrahim 3

1 Pathology, Forensic Medicine & Toxicology, Minufiya University, El-SadatCity Branch, Egypt2 Toxicology and Forensic Medicine Department, Faculty of VeterinaryMedicine, Cairo University, Egypt3Department, Faculty of Veterinary Medicine, Minufiya University, El-SadatCity Branch, EgyptE-mail address: [email protected] (A.M. Morgan).

Objectives: This work has been carried out to investi-gate the ameliorating effect of curcumin (80 mg/kgbt.w.P.O.) and/or zinc (227 mg/L in D.W) against NiCl2(1200 ppm in D.W.) - induced hepatotoxicity.

Methods: One hundred and twenty female albino ratswere divided into two groups (A & B), of 60 rats each and eachgroup further subdivided into 6 sub-groups of 10 rats each.Group A was administrated the antioxidants concomitantlywith nickel for 8 weeks; group B was administered the anti-oxidants two weeks prior to nickel exposure and 8 weeksconcomitantly with nickel. Sub-group A1 & B1 (Control), A2 &B2 (Ni), A3 & B3 (Niþ corn oil), A4 & B4 (Niþ curcumin), A5 &B5 (Ni þ Zn) and A6 & B6 (Ni þ curcumin þZn).

Results: Administration of NiCl2 alone to rats inducedsignificant elevation in serum ALT, AST, serum and hepaticrenal MDA level, and CAT activity. Also, NiCl2 induceda significant decrease in serum and hepatic GSH levels and

SOD activity. In addition, histopathological changes in liver(vacuolization of hepatocytes and increase in number ofkupffer cells) were also recorded. Curcumin and or/zincadministration improved the altered biochemical parame-ters and histological structures, especially in the pre-treated animals. The greatest improvement was seen withpre-treatment using both curcumin and Zn together.

Conclusions: Curcumin and Zn improved the biochem-ical parameters and histologic features in nickel-inducedhepatotoxicity in rats. Pre-treatment with curcumin and Zncombintion provided the greatest protection.

Keywords: nickel, hepatotoxicity, curcumin, zinc, antioxidants10.1016/j.toxicon.2012.04.139

J. Pharmacology

139. Comparison of Angiotensin Converting EnzymeInhibitors and Angiotensin II Type 1 Receptor Blockadefor the Prevention of Premalignant Changes in the Liver

Mahmoud A. Mansour 1, Hani Al-Ismaeel 1,Ammar C. Al-Rikabi 2, Othman A. Al-Shabanah 1

1Department of Pharmacology, College of Pharmacy, King Saud University,Riyadh 11451, Saudi Arabia2Department of Pathology, College of Medicine, King Saud University, Riyadh11461, Saudi ArabiaE-mail address: [email protected] (M.A. Mansour).

Methods: We investigate and compare the possibleantitumor activity of clinically used angiotensin convertingenzyme(ACE) inhibitors; captopril, perindopril and angio-tensin II type 1 receptor (AT1R) blocker, losartan againsthepatocarcinogenesis initiated by diethylnitrosoamines(DENA) and promoted by carbon tetrachloride (CCl4).Diethylnitrosamine (DENA) (200 mg/kg i.p.) initiated andcarbon tetrachloride (CCl4) (2 ml/kg i.p.) promoted hep-atocarcinogenesis in male Wistar rats after 8 weeks.

Results: Hepatocarcinogenesis was manifested biochemi-cally by elevation of serum hepatic tumor markers tested;a-feto protein (AFP) and carcinoembryonic antigen (CEA). Inaddition, hepatic carcinogenesis was further confirmed bya significant increase in hepatic tissue growth factors; vascularendothelial growth factor (VEGF) and basic fibroblast growth



Table 1Effects of ACE inhibitors and AT1R blocker administration on DENA-induced changes in rat hepatic growth factors; VEGF, TGF-b1 and FGF.

Groups VEGFpg/ml

TGF-b1pg/ml

FGFpg/ml

Control 144.43 � 3.06 132 � 26 1005þ56Captopril 142.8 � 802 87 � 13.5 865 � 81Perindopril 133.3 � 6.22 150 � 20 1024 � 41Losartan 122 � 4.4 124.4 � 17.7 1035 � 54DENA 210 � 5.08* 158.5 � 44.9 1444 � 52*DENAþ Captopril 129.2 � 5.04# 123.7 � 28.5 1149 � 57DENAþ PerindoprilDENAþLosartan

144.7 � 7.8#

128.13 � 3.9#169.8 � 18.774 � 10.4

1156 � 83#

1025 � 116#

All data represent mean values � SEM (n¼10). ACE inhibitors and AT1Rblocker were given in drinkingwater for 15 consecutive days before DENAadministration and continue during the experimental period.

* Significant difference from control group.# Significant difference from DENA group. P < 0.05.

Table 2Effects of ACE inhibitors and AT1R blocker administration on DENA-induced changes in rat hepatic MMP-2, TIMP-1 and hydroxyproline.

Groups MMP-2ng/ml

TIMP-1pg/ml

Hydroxy-prolineng/ml


factor (FGF). Moreover a marked increase in matrix metal-loproteinase-2 and hydroxyproline content were alsoobserved. Hepatocarcinogenesis was further confirmed bya significant decrease in hepatic endostatin and metal-lothonein level. Long-term administration of the selecteddrugs for 2 weeks before and throughout the experimentalperiod produced a significant protection against hepaticcarcinogenesis. The present results claimed that differentdoses of the selected drugs succeeded in normalization ofserum tumor markers. Furthermore, the drugs reduced theelevated level in the hepatic growth factors, matrix metal-loproteinase-2 and hydroxyproline induced by the hep-atocarcinogen. Moreover, the amelioration was alsoaccompanied by augmentation of hepatic content of metal-lothionein and endostatin. Histopathological examination ofliver tissues of rats treated with DENA–CCl4 correlated withthe biochemical observations.

Conclusions: These findings suggest a similar protectiveeffect of ACE inhibitors; captopril; perindopril and AT1Rblocker, losartan against premalignant stages of livercancer in the DENA initiated and CCl4 promoted hep-atocarcinogenesis model in rats. Therefore, RAS especiallyangiotensin II (Ang II) and AT1R interaction plays a pivotalrole hepatocarcinogenesis development.

Fig. 3. Liver from rat treated with DENA and CCl4 showing necrotic hepa-tocytes with occasional dysplastic nuclei (arrow head). The adjacent portaltracts were infiltrated by numerous inflammatory cells including manyeosinophils. H&E X 400.

Fig. 2. Effects of ACE inhibitors and AT1R blocker pretreatment before carcin-ogen intoxication onhepaticmetallothionein andendostatin. Bothweremeasureby ELISA kits. The results were expressed as ng/mL. Each column represents themean of 10 rats with a vertical bar showing the SEM. *Significant difference fromcontrol group (p<0.05). #Significant difference from DENA group (p<0.05).

Control 3.47 � 0.23 945 � 22 509 � 18Captopril 3 � 0.24 716 � 79 534 � 40Perindopril 2.4 � 0.47 852 � 118 547 � 56Losartan 3.3 � 0.44 753 � 50 500 � 40DENA 14 � 0.966* 1419 � 157 893 � 73*DENAþ Captopril 4.66 � 0.57# 1021 � 55 468 � 24#

DENAþ PerindoprilDENAþLosartan

5.3 � 0.57#

5.17 � 0.94#1856 � 147*1312 � 157

497 � 24#

621 � 32#

All data represent mean values � SEM (n¼10).ACE inhibitors and AT1R blocker were given in drinking water for 15consecutive days before DENA administration and continue during theexperimental period.

* Significant difference from control group.# Significant difference from DENA group. P < 0.05.

Keywords: losartan, captopril, perindopril10.1016/j.toxicon.2012.04.140

140. Identification, Pharmacological and Structuralcharacterization and Engineering of Three-finger Toxinsinteracting with GPCRs

Guillaume Blanchet 1, Gilles Mourier 1, Elodie Marcon 1,Bernard Gilquin 2, Nicolas Gilles 1, Denis Servent 11 Service d'Ingénierie Moléculaire des Protéines, Biologie Structurale etMécanismes, Gif-sur-Yvette, France2 Service de Bioénergétique, Biologie Structurale et Mécanismes.Gif-sur-Yvette, FranceE-mail address: [email protected] (D. Servent).

Background: To subdue their prey or protect themagainst predators, venomous animals have selected toxinsthat primarily interact with voltage-gated and ligand-gatedion channels, targets which play crucial roles in severalbiological functions. Nevertheless, some toxins are known



to recognize other molecular target families, such as theG-Protein Coupled Receptors (GPCRs). The first part of thetoxins interacting with GPCRs can be considered as func-tional mimetics of natural agonists of the receptor whileother toxins display structure and pharmacological profilesunrelated to any natural endogenous ligands.

Results: Here we first presented our last results relatedto the identification and pharmacological characterizationof new three-finger fold toxins interacting with alpha-adrenergic receptors. Two toxins: r-Da1a and r-Da1b, wereisolated from the Dendroaspis angusticeps snake venom andcharacterized for their high affinity and specific interactionwith a1a- and a2-adrenoceptors, respectively. The particularpharmacological profile of these toxins allows envisioningtheir exploitation as imaging or therapeutical agents, as forexample for r-Da1a in the treatment of benign prostatichyperplasia. In addition, structure-function studies ofvarious toxin-receptor complexes were performed inorder to identify at the molecular level the origin of thespecificity of these interactions. Our results reveal howMT7toxin recognizes the muscarinic M1 receptor and proposesa structural model of this complex in which the MT7interacts with a dimeric form of the receptor. This modelstructurally supports the high affinity and selectivity of theMT7-hM1 interaction, shows the atypical mode of interac-tion of this allosteric ligand with the GPCR receptor and canbe used as a starting point to design new ligands withpredetermine pharmacological property. Thus, MT7 engi-neering using block permutations lead to the generation oftoxins with new muscarinic/adrenergic functional profiles.

Conclusion:Our results identifymolecular determinantsinvolved in the affinity, selectivity and functional property ofaminergic toxins and highlight the ability of the three-fingertemplate to support various GPCRs interacting profiles.

References

C. Marquer et al. Structural model of ligand-G protein-coupled receptor (GPCR) complex based on experimentaldouble mutant cycle data: MT7 snake toxin bound todimeric hM1 muscarinic receptor, J Biol Chem 286 (2011)31661-31675.C. Rouget, et al. Identification of a novel snake peptide toxindisplaying high affinity and antagonist behaviour for thealpha2-adrenoceptors, Br J Pharmacol 161 (2010) 1361-1374.L. Quinton, et al. Isolation and pharmacological character-ization of AdTx1, a natural peptide displaying specificinsurmountable antagonism of the alpha1A-adrenoceptor,Br J Pharmacol 159 (2010) 316-325.

Keywords: three-finger toxins, GPCRs, bio-engineering10.1016/j.toxicon.2012.04.141

141. Lipid Bilayer Condition Abnormalities FollowingMacrovipera lebetina obtusa and Montivipera raddeiSnake Envenomation

Naira M. Ayvazian, Narine A. Ghazaryan, Lusine GhulikyanLaboratory of Toxicology, Institute of Physiology of NAS RA, Yerevan, ArmeniaE-mail address: [email protected] (N.M. Ayvazian).

Background: Viper bites are an endemic public healthproblem in Armenia, even in the cities. Venoms producedby snakes of the family Viperidae contain proteins thatinterfere with the coagulation cascade, the normal hae-mostatic system and tissue repair, and human envenoma-tions are often characterized by clotting disorders,hypofibrinogenemia and local tissue necrosis.

Methods: Studies on the interaction of snake venomand organized lipid interfaces have been conducted usinga variety of systems, including BLMs, SUVs and LUVs. Giantunilamellar vesicles (GUVs) with a mean diameter of 30 mmhave a minimum curvature and mimic cell membranes inthis respect. GUVs were formed from the total lipid fractionfrom bovine brain by the electroformation method (Ange-lova and Dimitrov, 1987). Macrovipera lebetina obtusa andMontivipera raddei venom was added to the samplechamber before the vesicles were formed. The membranefluorescence probes, ANS and pyrene, were used to assessthe state of the membrane and specifically mark thephospholipid domains. Fluorescent spectra were acquiredon a Varian fluoremeter instrument.

Results: The membrane fluorescence probes, ANS andpyrene, were used to assess the state of membrane andspecifically mark the phospholipid domains. Independent oftheir lipid composition, all GUVs modified by Macroviperalebetina obtusa venom were enlarged in size as venom-dependent lipid hydrolysis proceeded. In contrast, liposomesmodified with Montivipera raddei venom demonstrate a socalled “oval deformations” and venom-dependent shrinkingresponse. In addition to the visible morphological changes,ANS and pyrene also allows us to quantify the fluiditychanges in the membrane by measuring of the fluorescenceintensity. Thepresenceof vipervenom inGUVsmedia revealsa noticeable decreasing of membrane fluidity compare thecontrol, while the binding of fluorophores with GUVsmodified by venom lead to appearance of channel activity.

Conclusions: These studies emphasize the importanceof a membrane surface curvature for its interaction withenzymatic components of venom.

Keywords: BLM, GUV, electroporation, snake venomics, artificialmembranes10.1016/j.toxicon.2012.04.142

142. From alpha-Conotoxins and alpha-Neurotoxins toEndogenous “Prototoxins" and Binding Sites inNicotinic Acetylcholine Receptors

Victor I. Tsetlin, Yuri N. Utkin, Igor E. Kasheverov,Ekaterina N. LyukmanovaShemyakin-Ovchinnikov Institute of Bio organic Chemistry, Russian Academyof Sciences, Moscow, RussiaE-mail address: [email protected] (V.I. Tsetlin).

Background: Snake venom alpha-neurotoxins helpedto isolate a muscle-type nicotinic acetylcholine receptor(nAChR) and more recently the acetylcholine-bindingprotein (AChBP), an excellent model for the ligand-bindingdomains of all nAChR subtypes. Alpha-conotoxins fromConus snails are more selective in distinguishing neuronal




nAChRs. A breakthrough was the discovery of endogenous“prototoxin “ Lynx1, belonging to the same family of three-finger proteins as snake neurotoxins, attached to themembranes via glycosyl phosphatidylinositol (GPI) anchornear nAChRs and modulating their function. The reportcovers our recent findings, including those in collaborationwith European laboratories.

Methods: HPLC analysis of snake venoms, primarystructure determination, NMR and X-ray for 3D-structures,E. coli heterologous expression of three-finger proteins ,solid-phase synthesis of alpha-conotoxins. Computermodeling, molecular dynamics , X-ray analysis, electro-physiology and radioligand analysis for theoretical andexperimental studies of alpha-conotoxins and othercomplexes with AChBPs and nAChRs.

Results: Novel alpha-conotoxin PnIA analogs and radi-oiodinated derivatives having higher affinity for alpha7nAChRs and L. stagnalis AChBP were designed and synthe-sized (Kasheverov et al, Mar. Drugs, 2011). Three-fingerproteins with an additional disulfide in the loop I, namelyWTX Naja kaouthia and water-soluble domain of Lynx1(ws-lynx1) were expressed in E. coli and shown to act onnAChRs and muscarinic receptors; ws-lynx1 was demon-strated to bind at the classical site for agonists/competitiveantagonists at AChBPs and muscle-type nAChRs, butbeyond it at neuronal nAChRs (Luykmanova et al, Bio-khimia, 2009; J. Biol. Chem, 2011; Mordvintsev et al, FEBS. J.2009). A covalent dimer of alpha-cobratoxin, interactingwith alpha3beta2 nAChRs, was discovered in Naja kaouthiavenom and its X-ray structure recently determined (Osipovet al, J. Biol. Chem. 2008, 2012).

Discussion: We demonstrated the advantages ofcombined approach utilizing novel snake venom proteinneurotoxins and synthetic alpha-conotoxins and theiranalogs as tools in nAChR research. These instruments aresupplemented by theoretical and experimental approachesrevealing the structure of AChBP complexeswhich providesinformation about the binding sites in distinct nAChRsubtypes. In particular, with our participation it wasdemonstrated that binding of alpha-conotoxins (antago-nists) produces conformational changes opposite to thoseinduced by agonists (Celie et al, Nature Str. & Mol. Biol.,2005), while for such antagonists as d-tubocurarine andstrychnine different orientations are possible within thesame AChBP molecule (Brams et al, PloS Biology, 2011).

Conclusion: Peptide and polypeptide neurotoxinsprovide valuable information about the binding sites indistinct muscle-type and neuronal nAChRs shedding lighton functional mechanisms and providing basis for drugdesign.

Keywords: three-finger toxins, alpha-conotoxins, nicotinic acetylcholinereceptors10.1016/j.toxicon.2012.04.143

143. Exploring ‘Labyrinth’ of Pain with Scorpion ‘Sting’

Liu Zhirui, Ji YonghuaLaboratory of Neuropharmacology and Neurotoxicology, ShanghaiUniversity, Shanghai, ChinaE-mail address: [email protected] (J. Yonghua).

Background: Voltage-gated sodium channels (VGSCs),responsible for initiation and probagation of action poten-tials in most excitable cells, are implicated to play a criticalrole in the development and maintenance of pain observedin primary afferent neurons following nerve and tissueinjury. Scorpion venemation is a severe medical problem inmany regions across the world. Victims of envenoming bya scorpion suffer a variety of pathologies, such as fever,convulsion and intense pain.

Discussion: Although the common mechanisms of thispathological pain symptoms have been deduced in partover the years, detail information like pain transmission,integral processing and therapeutic intervention andcontrol have not yet been fully worked out. Currently, it hasbeen demonstrated that the toxic components in scorpionvenom are mainly neurotoxic peptides targeting on variousion channels including VGSCs at large.

Conclusions: This review will present a state of the artprogress in studying the underlying behavioral phenotypesand peripheral mechanisms of pain responses mimicing bya novel experimental BmK (Buthus martensi Karsch) scor-pion sting pain model. Through looking in-depth explora-tion of venom composition, pharmacological bindingcharacteristics, intra-/extracellular electrophysiologicalactivities and molecular determinants, a possible cross-talknetwork regarding the VGSCs-mediated pain initiation andmaintanence in overall neuronal sensing and responsespathway by scorpion sting could be approached.

Keywords: pain, scorpion sting, BmK, voltage-gated sodium channels10.1016/j.toxicon.2012.04.144

144. Analgesic Effect of Crotalphine in a New Model ofRat Bone Cancer Pain

Yara Cury 1, Vanessa P. Gutierrez 1, Patricia Brigatte 2,Vanessa O. Zambelli 1, Gisele Picolo 1, Juliana S. deCarvalho 1, Fabio Marques 3

1 Laboratorio Especial de Dor e Sinalizacao, Instituto Butantan, Sao Paulo,Brazil2Universidade Estadual de Sao Paulo (UNESP), Rio Claro, Brazil3Hospital das Clinicas, Universidade de Sao Paulo, Sao Paulo, BrazilE-mail address: [email protected] (Y. Cury).

Background: Crotalphine (CRP), a peptide first identi-fied and isolated from the South American rattlesnakeCrotalus durissus terrificus venom, induces analgesic effectmediated by opioid receptors. The aim of this work is tocharacterize the analgesic effect of crotalphine in a newmodel of bone cancer pain induced by inoculation ofWalker 256 tumor cells into the rat femoral cavity.

Methods: Bone tumor implantation and metastasiswere determined by histopathological analysis. Bonemetabolic alterations were determined by scintigraphy,using 99mTc-MDP. Femoral images were obtained beforeand 7,14 and 21 days after tumor cell injection. Bone cancerpain was characterized by the presence of hyperalgesia (ratpaw pressure test) and allodynia (von Frey filaments).

Results and Discussion: Photomicrographs analyzed21 days after injection of tumor cells, demonstrated thepresence of tumor cells in the femur of the animals.




Incorporation of 99mTc-MDPwas significant 7,14 and 21days,suggesting the development of tumor on the femoral cavity.Histopathological analysis demonstrated the presence oftumor cells in the lung and spleen, but not in the liver andkidneys of the rats. The results indicate that cells inoculatedinto femoral bone marrow can spread to some organs,including lymphoid organs. Hyperalgesia and allodyniaweredetected on days 1, 3, 7, 14 and 21 after cell inoculation.Interestingly, the paw withdrawal threshold in the von Freytest was reduced not only in the ipsilateral hind paw but alsoin the contralateral one, demonstrating the existence ofbilateral allodynia (mirror-image pain). To evaluate theinvolvementof prostanoids in these nociceptive phenomena,Indomethacin, a cyclooxygenase inhibitor, was administered3,7,14 and 21 days after tumor cell injection. Indomethacinonly partially inhibited hyperalgesia and allodynia inducedby bone cancer, indicating the involvement of prostanoids inbone cancer pain. The contribution of prostanoids is moresignificant within the first 3 days after cell injection. CRP(8mg/kg) administered on day 21, blocked hyperalgesia,allodynia and mirror image pain. The analgesic effect wasdetected up to 2 days after peptide administration and wasblocked by k-opioid receptor antagonist and partiallyinhibitedby d-opioid antagonists, indicating the involvementof opioid receptors. Morphine only partially inhibited allo-dynia and hyperalgesia.

Conclusions: Results indicate that injection of tumorcells causes bone cancer and pain. CRP induces a potent andlong-lasting antinociception in this model, with higherefficacy as compared to standard analgesic drugs.

Acknowledgments:FAPESP, CAT/CEPID/FAPESP and INCTTOX.

Keywords: crotalphine, analgesia, bone cancer pain10.1016/j.toxicon.2012.04.145

145. In vitro Vascular Activity of Crude Bungaruscandidus and Bungarus fasciatus Crude Venoms

Muhamad Rusdi Ahmad Rusmili 1,2, Iekhsan Othman 2,Mohd Rais Mustafa 3, Wayne Hodgson 1

1Monash Venom Group, Department of Pharmacology, Faculty of Medicine,Nursing and Health Sciences, Monash University, Clayton Campus, Victoria,Australia2 Jeffery Cheah School of Medicine and Health Sciences, Monash University ,Sunway Campus, Bandar Sunway, Selangor, Malaysia3Department of Pharmacology, Faculty of Medicine, University of Malaya,Kuala Lumpur, MalaysiaE-mail address: [email protected] (W. Hodgson).

Background: The South East Asian region containsa large number of species of venomous snakes that havenot been extensively studied. Bungarus candidus (Malayankrait) and Bungarus fasciatus(Banded krait) are two medi-cally important snakes in this regionwith highly neurotoxicvenoms. Even though considerable work has been done onthe neurotoxicity of the venoms, not much is known aboutother activities of these venoms.

Methods: In this study, we examined the in vitrovascular activity of Bungarus candidus and Bungarus fas-ciatus venoms using isolated rat thoracic aorta.

Results: Both venoms were found to produce dose-dependent relaxation (1-50mg/ml) in precontracted endo-thelium-intact aorta but not endothelium-denuded aorta.The relaxation effect by both venoms was partially inhibi-ted by the cyclooxygenase inhibitor indomethacin(5mM).The muscarinic receptor antagonist atropine (1mM)only partially inhibited the relaxation response producedby Bungarus candidus but not Bungarus fasciatus. However,the nitric oxide synthase inhibitor L-NOLA (100mM)and pre-treatment of the venom with 4-bromophenacylbromide (1.8mM) inhibited the relaxation responseproduced by both venoms. Preliminary screening on thefour fractions obtained by size-exclusion chromatographyindicated that the relaxation effect was produced bya single fraction. LCMS/MS analysis of this fraction indi-cated that it consisted mainly of phospholipase A2 toxins.

Conclusions: These data suggest that vasodilatorphospholipase A2/cyclooxygenase metabolites and endo-thelium-derived nitric oxide may play important roles inthe venoms vascular activity.

Keywords: Bungarus candidus, Bungarus fasciatus, blood vessel10.1016/j.toxicon.2012.04.146

146. An Insecticidal Spider Toxin that Acts as a PositiveAllosteric Modulator of Insect Nicotinic AcetylcholineReceptors

Monique J. Windley 1, Glenn F. King 2,Graham M. Nicholson 1

1 School of Medical & Molecular Biosciences, University of Technology,Sydney, NSW, Australia2 Institute for Molecular Bioscience, University of Queensland, Brisbane, QLD,AustraliaE-mail address: [email protected] (G.M. Nicholson).

Background: k-Hexatoxins (formerly k-atracotoxins)are a family of excitotoxic insect-selective neurotoxinsfrom Australian funnel-web spiders (Hexathelidae: Atra-cinae) that are lethal to a wide range of agronomically andmedically relevant insects, but display no toxicity towardsvertebrates. The prototypic k-HXTX-Hv1c blocks nativeand heterologously expressed (pSlo) cockroach BKCachannels, but not cockroach NaV or CaV channels ormammalian BKCa channels. Despite the high affinity andselectivity of k-HXTX-Hv1c for insect BKCa channels, wehave recently discovered that the insect BKCa channel doesnot appear to be the lethal target of the toxin. The aim ofthis study was therefore to determine the lethal target ofk-hexatoxins.

Methods: Actions of k-HXTX-Hv1c on voltage- andtransmitter-gated ion channels were determined usingwhole-cell patch clamp analysis of dorsal unpaired median(DUM) neurons from the American cockroach Periplanetaamericana. Acute toxicity tests were performed in crickets(Acheta domesticus) by intrathoracic injection.

Results: Acute toxicity tests revealed that the BKCa

blockers paxilline, charybdotoxin and iberiotoxin, which allblock insect BKCa channels with IC50 values < 30 nM, arenot lethal in crickets. This suggests that k-HXTX-Hv1c hasadditional molecular targets important for its lethality.




Subsequent testing of cockroach KV channels revealed thatk-HXTX-Hv1c failed to significantly block cockroach KNaand KDR channels, but 1 mM k-HXTX-Hv1c did cause a 20%block of ‘A-type’ (KA) currents. This suggests that k-HXTX-Hv1c additionally targets either insect KV1 and/or KV4channel subtypes. In support, the non-selective KA blocker4-aminopyridinewas lethal in insects. However themodestactions at such high concentrations would indicatea different lethal target. Accordingly, we assessed theactions of k-HXTX-Hv1c on insect GABA (GABAAR), gluta-mate (GluClR) and nicotinic acetylcholine (nAChR) recep-tors in DUM neurons. We found that 1 mM k-HXTX-Hv1cfailed to significantly affect GABAARs but did producea modest 21% increase in GluClR currents. In contrast,k-HXTX-Hv1c produced a concentration-dependent slow-ing of nicotine-evoked nAChR current decay (EC50 ¼ 200nM) and reversed nAChR current desensitisation. Theseactions occurred without any alterations to the amplitudeof nAChR currents or the concentration-response curve tonicotine. These findings are consistent with a positiveallosteric modulation of nAChRs.

Conclusion: k-HXTX-Hv1c represents the first peptidetoxin that selectively modulates insect nAChRs witha mode of action similar to the excitotoxic insecticide spi-nosyn A. Given the similar phenotype and mechanism ofaction, we propose that the likely lethal target of k-HXTX-Hv1c is the insect nAChR.

Keywords: k-hexatoxins, nicotinic acetylcholine receptor, insecticidal,spider toxin, ion channels, positive allosteric modulator10.1016/j.toxicon.2012.04.147

147. Isolation and Pharmacological Characterisation ofNeurotoxins from the Venom of Three Species ofAustralian Copperheads (Austrelaps spp.) and theEfficacy of Tiger Snake Antivenom to Prevent or ReverseNeurotoxicity

Francesca Marcon 1, Mathieu Leblanc 2, Pierre Escoubas 2,Graham M. Nicholson 1

1 School of Medical & Molecular Biosciences, University of Technology,Sydney, Australia2VenomeTech, Valbonne, FranceE-mail address: [email protected] (G.M. Nicholson).

Background: The venom of the Australian lowlandscopperhead (A. superbus) produces significant, and poten-tially lethal, neurotoxic paralysis in cases of clinical enve-nomation. However, little is known about the neurotoxiccomponents within this venom or the venoms of therelated alpine (A. ramsayi) or pygmy (A. labialis) copper-heads. The aim of this study was to isolate and characterizethe neurotoxins responsible for these neurotoxic actions atthe skeletal neuromuscular junction.

Methods: Neurotoxins were purified using size-exclusion, cation-exchange and reverse-phase liquidchromatography. Masses and primary sequences weredetermined using ESI- and MALDI-TOF mass spectrometrycombined with enzymatic cleavage and Edman degrada-tion. Purified neurotoxins were pharmacologically char-acterised using the chick biventer cervicis nerve-muscle

preparation to determine their site and mechanism ofaction.

Results: Similar to an expanding number of Australianelapid snakes, all three species displayed potent presynapticneurotoxicity. This was due to the presence of a slow-acting,high molecular mass (> 40 kDa), multimeric snake presyn-aptic PLA2 neurotoxin (SPAN) complex within each venom.Further characterisation of the A. superbus SPAN complex, P-EPTX-As1a, revealed classical triphasic alterations to neuro-transmitter release and fade in tetanic tension. Interestingly,two of the subunits of P-EPTX-As1a displayed high sequencehomology with the a- and b-subunits of the SPAN complex,taipoxin, from the Coastal taipan (Oxyuranus scutellatus), anunrelated Australian elapid. Importantly, the activity of allthree SPAN complexes could be prevented, but not reversed,by CSL monovalent tiger snake antivenom (TSAV). Muscleparalysis also resulted from the activity of a number ofpostsynaptic a-neurotoxins present in all three venoms. Inthe case ofA. labialis venom, themost potent activitywas dueto a-EPTX-Al2a (8074 Da), a long-chain a-neurotoxin.a-EPTX-Al2a causes potent pseudo-irreversible antagonismat the skeletal muscle nicotinic acetylcholine receptor (pA2¼7.902 � 0.002). a-EPTX-Al2a contains five disulphide bondsand shares both significant sequence homology and phar-macophore residues of classical long-chain postsynaptic a-neurotoxins. Of clinical importance was the finding that thepostsynaptic neurotoxic actions of a-EPTX-Al2a can be pre-vented, but only partially reversed, by TSAV.

Conclusion: The venoms of all three Austrelaps spp.exhibit potent presynaptic and postsynaptic neurotoxicitythat may contribute to the clinical picture of envenomation.This is the first study to validate the use of TSAV in cases ofclinical envenomation by Austrelaps spp. Nevertheless, ithighlights the importance of not delaying administration ofantivenom, once neurotoxicity becomes evident, due to thelack of antivenom efficacy against presynaptic and irre-versible postsynaptic neurotoxins.

Keywords: Austrelaps, Australian copperhead, snake presynaptic PLA2neurotoxin, postsynaptic a-neurotoxin, snake antivenom, neurotoxicity,neurotransmitter release, skeletal muscle nicotinic acetylcholine receptor10.1016/j.toxicon.2012.04.148

148. Characterization of Inflamin from Aipysuruseydouxii: A Novel Class of Toxin that InducesInflammation

Bhaskar Barnwal 1, R. Manjunatha Kini 1,21Department of Biological Sciences, National University of Singapore,Singapore2Department of Biochemistry and Molecular Biophysics, Medical College ofVirginia, Virginia Commonwealth University, Virginia, USAE-mail address: [email protected] (R.M. Kini).

Background: Aipysurus eydouxii (Marbled sea snake)belongs to the Hydrophiidae family. Venom of this snakehas not been extensively studied probably due to lowvenom yield. We sequenced the cDNA clones from itsvenom gland library and identified several novel proteins.Here, we describe the characterization of a novel protein,named as inflamin.




Methods: Inflamin has 94 residues including sixcysteines. We have recombinantly expressed inflamin inE.coli cells. Protein was refolded using redox buffer andpurified by RP-HPLC. The disulfide linkages were deter-mined by partial reduction and cyanylation-inducedchemical cleavage. We examined its effects in micethrough i.p. injections. We determined the production ofprostaglandins and prostacyclins in the peritonealexudate at various time points. Edema was monitoredafter intraplantar injection in rats. The mechanism ofaction of inflamin was studied by its effect on cPLA2 andCOX enzymatic activity in RAW264.7 macrophages.Western blotting was also performed to determine theexpression level of these enzymes.

Results: Refolded inflamin showed I-III, II-IV, V-VIdisulfide connectivity. In mice it induced writhing, which isprobably due to inflammatory pain. Indomethacin sup-pressed inflamin-induced writhing. Intraplantar injectioninduced inflammation and edema in rat paws. The perito-neal exudate showed the dose-dependent increase inprostaglandins. Levels of prostaglandins (PGF1a, PGE2,PGD2, PGF2a and TXB2) were highest after 10 min and thensubsided. The production of these prostaglandinsdecreased significantly upon indomethacin pre-treatment.We examined its mechanism of action on culturedRAW264.7 macrophages. At sub-cytotoxic concentration ofinflamin, the cPLA2 enzymatic activity increased within 5min of protein administration and becomes 150% comparedto saline after 10 min. Western blotting showed that cPLA2was phosphorylated at Ser505within 5min. COX-2 enzymeis induced after 3h which appears to start the second waveof prostaglandin production.

Discussion: The novel protein inflamin induces edemaand inflammatory writhing in experimental animals. Thisinflammation is due to the release of prostaglandins.Inhibition of COX enzyme by indomethacin decreasedprostaglandin production and writhing. Inflamin treat-ment leads to phosphorylation of cPLA2 and increase in itsactivity and the production of pro-inflammatoryeicosanoids.

Conclusions: Inflamin is a novel snake venom pro-tein that induces inflammation and pain. SinceA. eydouxii depends on eggs-only diet, we speculate thatthis toxin may be used as a defensive weapon againstpredators.

Keywords: snake venom, inflammation, prostaglandins10.1016/j.toxicon.2012.04.149

149. The Role of the Lymphatic System in the Absorptionof Micrurus fulvius Venom

Dayanira Paniagua 1, Lucía Jiménez 1, Camilo Romero 2,Irene Vergara 1, Arlene Calderón 1, Melisa Benard 1,Michael Bernas 3, Carlos Sevcik 4, Marlys Witte 3,Leslie Boyer 5, Alejandro Alagón 1

1 Instituto de Biotecnología, Universidad Nacional Autónoma de México,Cuernavaca, México2Departamento de Producción Agrícola y Animal, Universidad AutónomaMetropolitana, Unidad Xochimilco, México, DF, Mexico3Department of Surgery, University of Arizona, Tucson, AZ, USA

4 Instituto Venezolano de Investigaciones Científicas (IVIC), Caracas,Venezuela5Venom Immunochemistry, Pharmacology, and Emergency Response (VIPER)Institute, University of Arizona, Tucson, AZ, USAE-mail address: [email protected] (D. Paniagua).

Background: Coral snakes have short fixed fangsand a poorly developed system for venom delivery, therebyrequiring a chewing action to inject the venom; thisphysiology suggests that inoculation of the venom is bysubcutaneous (SC) route. Venom components inthe interstitial space must then pass to blood either directlyor through absorption to lymph for systemic distribution.

Methods: The contribution of lymph to the absorptionand systemic bioavailability of M. fulvius venom after SCadministration was determined using a central lymph-can-nulated sheep model. As the reference, we also used non-cannulated animals. Also, the kinetics of the venomwas fol-lowed after intravenous bolus injection. Each of the threegroups included four sheep; experiments lasted six hours andthe animals were kept under anesthesia during the wholeexperiment. Venom concentrations in serum and lymphwere measured by sandwich enzyme-linked immunoassay.

Results: Venom injected into blood stayed in systemiccirculation for 72.8�5 min (MRT), with a half-life (t1/2) of25.3�3.2min and a volume of distribution (Vss) of 11�0.8%.In contrast, when administered subcutaneously the venomMRT, t1/2 and Vss increased 5.5, 9 and 5.6-fold, respectively.The absorption of venom to blood was incomplete whenthe venomwas injected SC, with a recovery of 63�5% of theinitial dose during the 6 hours of the experiment. Lymphcontributed with 39% of the absorbed venom to blood.

Discussion: The low Vss of venom after IV administra-tion shows that tissues do not retain venom significantlyand that the steady state observed after SC injection is theresult of a slowabsorption process. The kinetics of venom inblood of the thoracic duct cannulated versus non-cannu-lated groups show that lymphatic absorption contributes tomaintain a prolonged steady state in systemic circulation.

Conclusions: These results show that the limitingprocess in the pharmacokinetics of SC injected M. fulviusvenom is its absorption and that the lymphatic systemplays a key role in the process. Supported by CONACyT(C382-08) and Instituto Bioclón S.A. de C.V.

Keywords: Micrurus fulvius, lymphatic system, absorption10.1016/j.toxicon.2012.04.150

150. Tailoring the Selectivity of Anuroctoxin for Kv1.3KD Channels

Ádám Bartók, Zoltán Varga, György PanyiUniversity of Debrecen, Department of Biophysics and Cell Biology, Debrecen,HungaryE-mail address: [email protected] (G. Panyi).

Background: The voltage-gatedKv1.3 potassium channelplays a key role in the activation of T lymphocytes by main-taining a negative membrane potential. By blocking thesechannels the proliferation of Tcells can be inhibited. This canresult in immunosuppression, which has a great potential inthe therapy of certain autoimmune diseases. Anuroctoxin,




a 35-amino-acid peptide isolated and characterized previ-ously from the venom of the scorpion Anuroctonus phaio-dactylus, blocks Kv1.3 with high affinity (Kd ¼ 0.7 nM).Although with lower affinity, the toxin blocks another Kþ

channel, Kv1.2 (Kd ¼ 6 nM), which is expressed in neurons,heart and smoothmuscle cells, thus this property of the toxinis not advantageous considering its potential clinical use.

Methods: We examined four variants of anuroctoxinproduced by solid-phase synthesis. Besides the wild-typetoxin we tested three mutant toxins designed by sequencealignment of published toxins selective for Kv1.2 or Kv1.3. Ourexpectation was that the mutations would improve theselectivity of the toxin for Kv1.3 over Kv1.2 keeping the highaffinity for Kv1.3.We investigated the effect of the toxinswithwhole-cell patch-clamptechniqueonactivatedT lymphocytesendogenously expressingKv1.3 channels and cells transfectedwith the gene of hKv1.1, hKv1.2 and IKCa1 channels.

Results: The effect of synthetic wild-type anuroctoxinwas similar to that of the natural toxin (Kv1.3: Kd ¼ 0.3 nMand Kv1.2: Kd ¼ 5.3 nM) proving the success of solid-phasesynthesis. The first mutant F32T was designed to examinethe role of the essential dyad in selectivity. F32T practicallylost affinity for Kv1.2 but also showed a slight decrease inaffinity for Kv1.3 (Kd ¼ 7.5 nM). We designed the mutantN17A in an attempt to increase affinity for Kv1.3. However,this affinity did not change significantly (Kd ¼ 0.9 nM) anda slight improvement in selectivity could be observed (Kv1.2:Kd¼ 18.9 nM). Combining the twomutations in one toxinweconstructed the mutant N17A,F32T where the advantageouseffect of both mutations could be detected. The N17A,F32Tmutant is highly selective (no significant effect on Kv1.2 at100 nM) and a high affinity blocker of Kv1.3 (Kd ¼ 0.6 nM).

Conclusions:With targeted mutations we designed andproduced a selective and high affinity blocker of Kv1.3. Ourresults provide the foundation for the possibility of theproduction and future therapeutic application of addi-tional, even more selective toxins.

Keywords: Kv1.3, scorpion toxin, selectivity10.1016/j.toxicon.2012.04.151

151. Immuno-Inflammatory Response after ScorpionEnvenomation: Potential Role of Eïcosanoids andHistamine H1-Receptor

Sonia Adi-Bessalem1,2, Amina Ladjal-Mendil 1,2,Djelila Hammoudi-Triki 1,2, Fatima Laraba-Djebari 1,21University Sciences and Technology Houari Boumediene, LaboratoryCellular and Molecular Biology, Faculty Biological Sciences, Algiers, Algeria2 Pasteur Institute in Algeria, Algiers, AlgeriaE-mail address: [email protected] (S. Adi-Bessalem).

Background: After scorpion envenoming, the massiverelease of neurotransmitters and inflammatory modulatorsis mainly due to neurotoxic components of scorpionvenoms. This release of mediators leads to a cascade ofpathological events, mainly in the cardio-respiratorysystem, by mechanisms which are not yet well understood.

Methods: The potential role of Histamine H1-Receptorand eicosanoids was investigated in mice using Histamine

H-1 antagonist (Prometazine, 5 mg/kg) or phospholipaseA(2) inhibitor (dexamethasone, 5 mg/kg) prior to theenvenomation with Androctonus australis hector, (Aah)venom. Inflammatory markers were measured in sera andtissue homogenates of envenomed mice.

Results: The results obtained showed that the inflam-matory process induced by this venom is characterized byhyperleukocytosis associated with elevated production ofnitric oxide and pro-inflammatory cytokines in peripheralblood. The migration of neutrophils and eosinophils inlungs and liver was confirmed by the release of myelo-peroxidase and eosinophil peroxidase. Hypoalbuminemiaand hyper-gammaglobulinemia accompanied by a signifi-cant activation of complement system were also thepredominant abnormalities observed after envenomation.Our results showed also that Aah venom induced signifi-cant increase in tissue lipid peroxidation, concomitantwith depletion of antioxidants. Histological analysisrevealed the presence of edema, hemorrhage andneutrophil infiltration in lung and liver tissue. Theleukocytosis, nitric oxide release and cellular peroxidaseactivities were inhibited by previous treatment withcorticosteroid (dexamethasone). However, fluid accumu-lation in the organs and tissue damage seem to be partiallyreduced. Administration of promethazine inducesa significant decrease of edema in liver and lung. Themyeloperoxidase and eosinophil peroxidase activitiesdecreased also significantly. Histological analysisconfirmed the inhibition of edema forming and inflam-matory cell recruitment in liver and pulmonary paren-chyma compared to envenomed mice. However, high lipidperoxidation rates and lower antioxidative protectionwere found in the liver and lung homogenates even afterinhibition histamine H-1receptor or eicosanoid mediators.

Discussion: Inflammatory response induced by scorpionvenom could be mediated by multiple mediators includinghistamine via H1-receptors. Dexamethasone pretreatmentmay suppress NF-kB activation and prevent the exacerbationof the inflammatory response after scorpion envenomation.

Conclusions: These results could help to provide saferand selective therapies in the complicated cases of enve-nomed patients. The use of antioxidants after scorpionenvenomation could be useful to produce a protectiveeffect against deleterious manifestations and damagesinduced by the scorpion venom.

Keywords: inflammation, eicosanoids, histamine, H1-receptor, scorpionvenom10.1016/j.toxicon.2012.04.152

152. Effect of Crotoxin on Secretory Activity ofPeritoneal Macrophages Co-cultivated with Tumor Cells.Involvement of Formyl Peptide Receptors

Yara Cury 1, Edilene S. Costa 2, Odair J. Faiad 2, Rui Curi 3,Sandra C. Sampaio 2

1 Laboratorio Especial de Dor e Sinalizacao, Instituto Butantan, Sao Paulo,Brazil2 Laboratorio de Fisiopatologia, Instituto Butantan, Sao Paulo, Brazil3 Instituto de Ciencias Biomedicas, Universidade de Sao Paulo, Sao Paulo,BrazilE-mail address: [email protected] (Y. Cury).




Background: Crotoxin (CTX) inhibits tumor growth andmodulates the functions of macrophages. Macrophagesprovide a defense mechanism against tumor cells and twodistinct polarization states, M1 and M2, have beendescribed for these cells. In the beginning of tumorprogression, M1 macrophages release reactive nitrogen/oxygen intermediates, cytokines and lipoxygenase-derivedeicosanoids, which may contribute to tumor inhibition. Incontrast, during tumor development, the release of thesemediators by tumor-associated macrophages (M2 cells) isinhibited, contributing to tumor development. Based onthese evidences, the aim of this work is to evaluate ifinhibition of macrophage by CTX contributes to thedecrease in tumor cell growth caused by this toxin.

Methods: The effect of CTX on the activity of macro-phages co-cultivated with LLC WRC 256 tumor cells wasevaluated. To determine tumor cell growth inhibition,macrophages (2x105 cells), obtained from rat peritonealcavity, were incubated with CTX (0.3mg/mL) for 2 h at 37oC.After this time, the macrophages were co-cultivated in thepresence of LLC WRC 256 tumor cells (2x104), previouslyplated in 96-well culture dishes. To determine the levels ofcytokines and eicosanoids, macrophages (5x105) wereincubatedwithCTX (0.3mg/mL) for 2 h and then co-cultivatedin presence of the tumor cells (5x104). The levels of IL-1b,LXA4 and 15-epi-LXA4 in culture supernatants were deter-mined by ELISA. The release of nitric oxide (NO) was deter-mined by the quantification of nitrite in the supernatants ofcultured macrophages.

Results: Co-cultivation of CTX-treated macrophagestogether with tumor cells caused a 25% reduction of tumorcell proliferation. A 35% increase in NO production wasdetected 48 h after co-cultivation. A 273% increase in IL-1blevels was detected 12 and 24 h after co-cultivation.Moreover, an increase in the levels of LXA4 (35%) and15-epi-LXA4 (2.3 fold) was observed 24 and 48 h afterco-cultivation. Boc-2 blocked the stimulatory effect of CTXon macrophage asecretory activity and the inhibitory effectof these cells on tumor cell proliferation.

Discussion: Taken together, these results indicate thatCTX modifies the secretory activity of M2 cells, which maycontribute to the inhibitory action of the toxin on tumorgrowth. Activation of formyl peptide receptors seems toplay a major role in this effect.

Conclusions: These data confirm the actions of CTX ondefence mechanisms and open new perspectives for thedevelopment of novel substanceswith therapeutic properties.

Financial support: FAPESP (09/52330-9; 1998/14307-9), CNPq/PIBIC, PAP and INCTTOX program.

Keywords: crotoxin, macrophage, tumor cells10.1016/j.toxicon.2012.04.153

153. Biochemical and Pharmacological Characterizationof Three-Finger Neurotoxins from the Venom of EasternCoral Snake (Micrurus fulvius fulvius)

Chun Shin Foo 1, Selvanayagam Nirthanan 1,2,R. Manjunatha Kini 3,4, Peter T.H. Wong 1

1Department of Pharmacology, Yong Loo Lin School of Medicine, NationalUniversity of Singapore, Singapore

2 School of Medical Science, Griffith University Gold Coast Campus,Queensland, Australia3Department of Biological Sciences, Faculty of Science, National University ofSingapore, Singapore4Department of Biochemistry, Medical College of Virginia, VirginiaCommonwealth University, Richmond, Virginia, USAE-mail address: [email protected] (S. Nirthanan).

Background: Animal venoms are rich sources of phar-macologically active proteins and polypeptides that exhibitremarkably high potency and specificity for different bio-logical targets, making them excellent probes for studyingthe distribution and function of ion channels and receptors.Neurotoxins derived from animal venoms in particular areinvaluable tools for studying neurotransmission. Theprimary objective of this work is the identification, isola-tion and biochemical and pharmacological characterizationof novel three-finger neurotoxins from the venom ofEastern coral snake, Micrurus fulvius fulvius (M. f. fulvius).

Methods: Neurotoxic fractions were purified fromM. f. fulvius venom using a two-step chromatographicapproach guided by ex vivo pharmacological screening inisolated avian and mammalian nerve-skeletal musclepreparations. Electrospray-ionization mass spectrometrywas used to determine themolecular weight of the purifiedproteins. Amino acid sequence of the target neurotoxinwassubsequently determined by N-terminal sequencing byEdman degradation and circular dichroism spectroscopywas used to determine the secondary structure.

Results: Crude venom (1 - 100 mg/ml) produced time-and concentration-dependent inhibition of nerve-evokedtwitch responses in indirectly stimulated chick biventercervicis muscle with an IC50 of 4.5 mg/ml. Bioactivity-guided HPLC purification led to the identification of a 7 kDaneurotoxin, subsequently named MFTx2. Analysis of theamino acid sequence and secondary structure indicatedthat this is a short-chain three-finger neurotoxin whichproduced potent postsynaptic neuromuscular blockadewith an IC50 of 40 nM, that is comparable to erabutoxin-b.However, unlike the poorly reversible neuromuscularblockade produced by erabutoxin-b and other typical a-neurotoxins, MFTx2 produced rapid and near-completerecovery of nerve-evoked twitch responses upon washing.

Discussion: Postsynaptic neuromuscular blockade, asobserved by the abolishment ofmuscle contractile responsesto electrical nerve stimulation and to exogenous agonists,indicates that MFTx2 targets muscle nicotinic acetylcholinereceptors. Primary sequence comparison of MFTx2 andconventional curaremimetic a-neurotoxins revealed thatMFTx2 lacked many functionally important residues forinteraction with muscle-type nicotinic acetylcholine recep-tors, suggesting an alternate mechanism of reversible inter-action with postsynaptic nicotinic acetylcholine receptors.

Conclusions: A novel three-finger neurotoxin,MFTx2, which produced postsynaptic neuromuscularblockade with nanomolar affinity, was purified from M. f.fulvius venom. In contrast to typical curaremimetica-neurotoxins, the neuromuscular blockade produced byMFTx2 was rapidly and completely reversible.

Keywords: snake venoms, three-finger toxins, neurotoxins10.1016/j.toxicon.2012.04.154



154. First Pharmacological Study of the Venom of a RareAfrican Snake, Naja multifasciata duttoni

Alan L. Harvey 1, Edward G. Rowan 1,R. David G. Theakston 2, David A. Warrell 31 Strathclyde Institute of Pharmacy and Biomedical Sciences, University ofStrathclyde, Glasgow, UK2 Liverpool School of Tropical Medicine, Liverpool, UK3Centre for Tropical Medicine, University of Oxford, Oxford, UKE-mail address: [email protected] (A.L. Harvey).

Background: The burrowing cobra, Naja multifasciataduttoni is a rare snake that has hardly been studied becauseit is seldom found. Its venom has never been studiedscientifically. Two specimens were impounded by UKCustoms and transferred to the Liverpool School of TropicalMedicine, allowing the opportunity to collect and study thepharmacological effects of the venom of this species.

Methods: Venomwas milked from both snakes, pooledand dried. Aliquots were reconstituted immediately beforeexperiments. Effects were determined on the isolatednervemuscle preparation, the chick biventer cervicis, usingindirect nerve stimulation and contracture responses toacetylcholine, carbachol and KCl.

Results: The venom produced a concentration-depen-dent reduction in the twitch responses to nerve stimula-tion. Complete block was reached within 20 min with 30ug/ml, while 3 ug/ml took about 90 min to cause completeblock. After responses to indirect stimulation were abol-ished by venom, responses to the agonists were tested:there were no responses to acetylcholine or to carbachol,while there were responses to depolarization induced byKCl. Responses to KCl were reduced after exposure tovenom at 30 ug/ml and this concentration alos causeda slow contracture of the muscle preparations.

Conclusions: Venom of the burrowing cobra, Najamultifasciata duttoni has similar pharmacological effectson nerve-muscle preparations as classical cobra venoms.The effects are consistent with the presence in tehvenom of postsynaptic neurotoxins and cardiotoxins (or'cytotoxins').

Keywords: cobra venom, cardiotoxin, neurotoxin10.1016/j.toxicon.2012.04.155

155. Expression of two endoplasmic reticulum stressmarkers, GRP78 and GADD153, is involved in themechanism of action of the Amblyomin-X.

Ana Marisa Chudzinski-Tavassi 1, Jean G. Souza 1,2,Simone M. Simons 1, Carolina M. Berra 1, Renata F. Sato 3,Roger Chammas R 3, Katia L.P. Morais 1,2

1Biochemistry and Biophysics Laboratory, Instituto Butantan, São Paulo,Brazil2Departamento de Bioquímica, Universidade Federal de São Paulo, SãoPaulo, Brazil3Departamento de Radiologia e Oncologia, Faculdade de Medicina daUniversidade de São Paulo, São Paulo, BrazilE-mail address: [email protected] (A.M. Chudzinski-Tavassi).

Introduction: A cDNA library of the A. cajennense ticksalivary glands was constructed and used to identify a gene

encoding a Kunitz-type protease inhibitor. A recombinantprotein, named Amblyomin-X was over expressed in E. coli.The expressed protein is able to promote apoptosis inmurinerenal adenocarcinoma (RENCA), decreased proteasomalactivity and increased pool of poly-ubiquitinylated proteinsin some tumor cell lines, suggesting an endoplasmic retic-ulum (ER)-stress. However, the mechanistic effects of thisprotein are still unclear.Objective: Evaluate the involvementof ER-stress in tumor cells treated or not with Amblyomin-X.

Methods: To evaluate two markers of the ER-stress,GRP78 and GADD153, the genic expressionwas perfomed byABI 7500 Real Time PCR System (Life Technologies) usingforward and reverse specific genes primers and analysis forWestern Blot with specific monoclonal antibodies wereperformed.

Results: RENCA cells treated with Amblyomin-Xshowed a modest effect in genes related to ER-stress, butsignificant differences in protein expression was observed.

Conclusion: The results suggest that ER-stress isinvolved in this process pro-apoptotic of the Amblyomin-X.

Keywords: tick, amblyomin-X, ER-stress10.1016/j.toxicon.2012.04.156

156. Neuromuscular activity of Bothrops fonsecai SnakeVenom and its Neutralization by Commercial BothropicAntivenom

Rita de Cássia de Oliveira Collaço 1, Gildo Bernardo Leite 1,José Carlos Cogo 2, Stephen Hyslop 1, Thalita Rocha 3,Priscila Randazzo-Moura 4, Léa Rodrigues-Simioni 11Departamento de Farmacologia, Universidade Estadual de Campinas(UNICAMP), Campinas, SP, Brazil2Centro de Estudos da Natureza, Universidade do Vale do Paraíba (UNIVAP),São José dos Campos, SP, Brazil3 Laboratório Multidisciplinar de Pesquisa, Universidade São Francisco (USF),Bragança Paulista, SP, Brazil4Departamento de Ciências Fisiológicas, Pontifícia Universidade Católica(PUC), Sorocaba, SP, BrazilE-mail address: [email protected] (L. Rodrigues-Simioni).

Background: Bothrops venoms can produce markedlocal effects such as hemorrhage and necrosis. Some ofthese venoms also cause neuromuscular blockade invertebrate nerve-muscle preparations in vitro. In this work,we examined themyotoxicity and neurotoxicity of Bothropsfonsecai venom in mouse isolated phrenic nerve-dia-phragm preparations. We also investigated the ability ofcommercial equine bothropic antivenom to neutralize theneuromuscular activity.

Methods: Mouse extensor digitorum longus (EDL)preparations mounted in Tyrode solution (37 oC)were incubated with venom (3-300 mg/ml) or Tyrode solu-tion alone (control) for 120min, after which the tissues wereprocessed for histological analysis. Venom PLA2 activity wasmeasured colorimetrically. For neutralization experiments,venom (100 mg/ml) was preincubated for 30 min withbothropic antivenom at ratios of 5:1 and 5:2 (w/v).

Results: Bothrops fonsecai produced neuromuscularblockade in EDL preparations, with the time (min) for 50%paralysis by 3, 10, 30, 100 and 300 mg of venom/ml being84.6�6.5, 76.7�11.8, 65.3�11.9, 41.2�6.4 and 46.6�4.2 min,





respectively (mean�SD; n¼5-7; p<0.05 compared to controlpreparations). Histological analysis showed muscle fiberswith edema, hypercontraction and vacuolization that resul-ted in ghostfibers. The PLA2 activity of B. fonsecai venom (100mg) was 1.75�0.5 mM HCl/min compared to 0.85�0.4 mMHCl/min for Crotalus durrissus terrificus (South Americanrattlesnake) venom (100 mg; positive control; n¼5 each;p<0.05). Commercial antivenom did not significantly affectvenom (100 mg/ml)-induced blockade at the manufacturer’srecommended venom:antivenom ratio of 5:1 (74.6�8.6%blockade after 120 min; n¼5); partial protection wasobserved at a ratio of 5:2 (only 37.5�5.5% blockade after 120min; p<0.05; n¼5). VenomPLA2 activitywasnot inhibitedbycommercial antivenom (activities of 1.70�0.05mMHCl/minand1.3�0.13mMHCl/min for venom:antivenomratios of 5:1and 5:2, respectively; n¼3 each).

Discussion and Conclusions: These results indicatethat B. fonsecai venom is myotoxic and neurotoxic in EDLpreparations. These effects may be mediated by venomPLA2 activity. Commercial bothropic antivenom onlypartially neutralized the neuromuscular activity, possiblybecause this venom is not included in the pool used toimmunize horses.

Keywords: bothropic antivenom, myotoxicity, neurotoxicity, PLA2 activity.10.1016/j.toxicon.2012.04.157

157. Validation of an Analytical Method to MeasureNeutralizing Potency of Anti Loxosceles Plasma

A. Chávez-Méndez 1, C. Olvera 2, A. Alagón 2, L. Olguín 1

1 Instituto Bioclon S.A de C.V, DF, México2 Insituto de Biotecnología, UNAM, MéxicoE-mail address: [email protected] (A. Chávez-Méndez).

Background: According to the World Health Organiza-tion guidelines “for the production control and regulation ofsnake antivenom immunoglobulins”, a plasma used toproduce an antivenom should be checked on its neutralizingpotency prior to fractionation to ensure the quality of theantivenom. The validation of the method to measure theneutralizing potency was performed to ensure its reliability.

Method: We validated an analytical method previouslydeveloped to measure in BALB/c mice, the neutralizingpotency of an anti-Loxosceles plasma against recombinanttoxins ofmedical relevant Loxosceles species. To themethodvalidationwe evaluated different parameters defined in theInternational Conference of Harmonization guidance:accuracy, precision, inter-assay variation, reproducibility,specificity and selectivity.

Results and Discussion: The method is specific, selec-tive, accurate and precise with variation coefficient lessthan 20 % and was demonstrated its reproducibility inter-assay between different days and inter-analysts. This vali-dation demonstrated that the method is statisticallysuitable and may be used as quality control test by manu-facturers as part of plasma release for the production of theanti Loxosceles antivenom.

Conclusions: The method is reliable and complies theestablished acceptance criteria to be used as quality control

test through of the determination of neutralizing potencyin plasma to produce an antivenom.

Keywords: Loxosceles, neutralizing potency, validation, antivenom10.1016/j.toxicon.2012.04.158

K. Physiology

158. EqT II Causes Endothelial Cell Damage andIntracellular Ca2D Riske in ECV-304

Mitja Maru�zin, Miha �Su�ster�si�c, Jernej Sitar, Primo�z Humar,Miha Bartoli�c, Du�san �SuputUniversity of Ljubljana, School of Medicine, Inst. of Pathophysiology,Ljubljana, SloveniaE-mail address: [email protected] (D. �Suput).

Background: Equinatoxin II (EqT II) is one of the acti-noporins isolated from the sea anemone Actinia equinaL. After oligomerization it forms pores in lipid membranes.EqT II is lethal in rat, and the mechanism of this action hasbeen explained by vasoconstriction and cardio respiratoryarrest. Endothelium may be responsible for the vaso-constrictory action of EqT II, but the action of EqT II onendothelial cells has not been studied in detail. In this studythe effects of EqT II on endothelial-like ECV-304 cells aredescribed.

Methods: EqT II was a kind gift of P. Ma�cek andG. Anderluh. ECV-304 cells were cultured in Petri dishand incubated with 1 mM fura-2 AM for 30 min. Fura-2was then washed out and the cells were mounted ina 200ml chamber on the stage of an inverted Axiovert100 microscope (Zeiss, Germany) and exposed to EqT IIin 1 to 100 nM concentration. To determine the source ofcytosolic Ca2þ rise a separate set of experiments wasperformed on thapsigargin pre-treated ECV-304 cells andin Ca2þ free medium. Samples were excited alternativelywith 340 and 380nm light wavelength from PolychromeII monochromator (T.I.L.L. Photonics, Germany) forratiometric determination of intracellular Ca2þ. Emittedlight at 510nm (10nm band-pass) was collected byMicroMAX CCD camera (Princeton Instruments, USA),digitized and stored in the imaging system. Data wererecorded every 10 s (0,1Hz) with 250 ms acquisitiontime. ROIs were selected over the nucleus of cells(central ROI) and surrounding cytoplasm (peripheralROI). ROI outside cells served for backgroundsubtraction.

Results: Exposure of cells to EqT II triggered a rise ofintracellular Ca2þ. Thapsigargin pre-treatment causingdepletion of intracellular stores had no influence on theEqT II action. Elimination of Ca ions from the bathingmedium by substitution with EGTA abolished the effect ofEqT II.

Discussion: Results show that EqT II induced rise incytosolic Ca2þ can be explained by influx of Ca2þ form thebathing solution and cannot be attributed to the release ofintracellular Ca2þ stores. It is well known that intracellularCa2þ is a crucial second messenger, and it may be expectedthat the rise in intracellular Ca2þ may cause cell swelling



Fig. 1. Top – Changes in intracellular Ca2þ concentration induced by equinatoxin II. A) Increase in fluorescence ratio (solid line) of intracellular fura-2 in thecentral ROI after addition of EqT II (100 nM final concentration) in culture medium containing 2 mM Ca2þ ions (dots show þ/� standard deviations at themeasurement points, n ¼ 16). B) Increase in fluorescence ratio of intracellular fura-2 in the peripheral ROI after addition of EqT II (same conditions as in A). C)Pretreatment with 50 nM thapsigargin resulted in a small and transient increase – no increase in fluorescence ratio (solid line) of intracellular fura-2 wasobserved in the central ROI after addition of EqT II (100 nM final concentration) in culture medium containing 2 nM EGTA – chelating agent selective for Ca2þ ions(dots show þ/� standard deviation at the measurement point, n ¼ 13). Bottom – Consecutive images of ECV-304 loaded with fura-2 in medium containing 2mMCa2þ; D) before; E) immediately after and F) 15 min after addition of 100 nM EqT II. For illustration a peripheral (bright circle) and central (dark circle) ROI isshown in the first image. Note the intensity changes of nuclear regions (arrows) produced by EqT II application. 1–2 min after application of EqT II first blebs ofcellular membrane appeared.


and a release of vasoactive substances from endothelialcells. Both effects may contribute to the vasoconstrictoryaction of EqT II.

Conclusion: EqT II causes cytosolic Ca2þ rise in ECV-304cells leading to endothelial dysfunction.

Keywords: equinatoxin, endothelium, ECV 304, intracellular calcium,vasoconstriction10.1016/j.toxicon.2012.04.159

159. Synaptophysin Expression and Neurotoxic Effectsof Some Bothropic Venoms and Toxins

Thalita Rocha 1,2, Luis A. Ponce-Soto 3, Sérgio Marangoni 3,Maria Alice da Cruz-Höfling 2

1Universidade São Francisco (USF), Laboratório Multidisciplinar de Pesquisa,Bragança Paulista, SP, Brazil2Universidade Estadual de Campinas (UNICAMP), Departamento deHistologia e Embriologia, Instituto de Biologia, Campinas, SP, Brazil3Universidade Estadual de Campinas (UNICAMP), Departamento deBioquímica, Instituto de Biologia, Campinas, SP, BrazilE-mail address: [email protected] (T. Rocha).

Background: Snake venoms are a mixture of bioac-tive compounds, displaying a variety of pathologicaleffects, including myotoxicity and neurotoxicity. SomeBothropic venoms as from B. alternatus are myotoxic;however, toxin BaTx is neurotoxic. Other venoms asfrom B. marajoensis and its toxin Bmaj-9 are exclusivelyneurotoxic. In general, the identification of Bothropictoxins with neurotoxic activity has been made throughpharmacological approaches, being the molecularmechanism less explored. The aim of this study was toinvestigate by western blotting the expression of syn-aptophysin, a synaptic vesicle structural protein, which

takes part in the physiology of the neuromuscularjunction and allows identifying presynaptically-actingneurotoxins.

Methods: Mouse phrenic nerve-diaphragm (PND) andchick biventer cervicis (BC) preparations were isolated andsubmitted to conventional myographic, histological andwestern blotting techniques. PND were treated with B.jararacussu venom (100mg/mL) and BthTX-I (from B. jarar-acussu venom, 50mg/mL) and BC were treated with B.alternatus venom (200mg/mL), BmjeTX-I and BmjeTX-II(from B. marajoensis venom, 10mg/mL). All experimentswere maintained at 37oC until the total neuromuscularblockade. After these the tissue was frozen for morpho-logical analyses or homogenate for synaptophysin immu-nodetection by western blotting.

Results: In PND, B. jararacussu venom and BthTX-Icaused a total and irreversible neuromuscular blockade(86�13.4 and 30�6.5 min respectively; n¼5-6, p<0.05compared to Tyrode controls) as well observed in BCtreated with BmjeTX-I and BmjeTX-II (31.2�3.5 and30�8.1 min respectively; n¼7-8, p<0.05 compared toKrebs controls). B. alternatus did not induce completeneuromuscular blockade (even after 120 min incubation).Qualitative morphological analyses showed that themuscle damage caused by these venoms and toxins lea-ded the fibres to edema, hypercontraction, vacuolizationand, consequently, to ghost fibre. Immunoblotting datashowed synaptophysin expression in PND and BC controlspreparations, but preparations incubated with B. jarar-acussu venom, BthTX-I, BmjeTX-I and BmjeTX-II toxins didnot.

Discussion: Synaptophysin expression is related tosynaptic vesicle integrity and its absence in preparationsincubated with venoms/toxins is in conformity with the



effect of snake venoms/toxins that affects ACh release at thenerve terminal hence resulting in presynaptic neuromus-cular blockade, as per B. jararacussu venom, BthTX-I,BmjeTX-I and BmjeTX-II toxins.

Conclusions: These results evidenced that the neuro-toxic effects induced by these venom and toxins can berelated to the absence of intact synaptic vesicle at theneuromuscular junction.

Financial support: FAPESP (2011/00001-1); CNPq.

Keywords: myotoxicity, neurotoxicity, synaptic vesicles, neuromuscularjunction10.1016/j.toxicon.2012.04.160

160. Role of SVMPs, Matrikines and TLR4 in SnakeVenom Induced Edema and Inflammation

Jay W. Fox 1, Alexandra Rucavado 2, Teresa Escalante 2,Junho Kim1, José M. Gutiérrez 2

1University of Virginia School of Medicine, Department of Microbiology,Immunology and Cancer Biology, Charlottesville, VA, USA2University of Costa Rica, Facultad de Microbiologia, Instituto ClodomiroPicado, San José, Costa RicaE-mail address: [email protected] (J.W. Fox).

Background: Snake venom metalloproteinases havelong been known to be potent agents of extracellularmatrix and significant information has been gained interms of understanding the mechanistic basis for how suchdegradation leads to hemorrhage and tissue damage.However, little is known regarding whether the products ofECM degradation may also contribute to the pathophysi-ology of envenomation.

Methods: Both in vitro and in vivo experimentswere performed to determinewhether SVMPdegraded ECMcould induce edema and inflammation. Additional studies toelucidate the mechanism of action were also undertaken.

Results: Venom SVMPs were demonstrated to degradeECM to produce matrikines which could induce edema andinflammation in vivo and up-regulate chemokines in tissueculture. Antagonism of the toll-like receptor 4 (TLR4) couldattenuate these activities.

Discussion: It has been recognized that someECM proteolytic products can activate TLR4 receptors toproduce edema and inflammation in certain diseases. Theability of SVMPs to similarly produce ECM matrikines tocontribute to the venom-induced edema and inflammationassociated with envenomation is a significant novel findingwhich in turn suggests additional therapeutic routes toattenuate morbidity and dead associated with snakeenvenomation.

Conclusions: SVMP degraded ECM products can func-tion as matrikines to contribute to envenomation patho-physiology via a matrikine-TLR4 mediated pathway toinduce edema and inflammation in the prey. These studiessuggest a more complex, nuanced role for SVMPs in venominduced pathophysiology than previously recognized andthat novel approaches to therapeutic treatment of

envenomation focused on this pathway may attenuateedema and inflammation associatedmorbidities in patients.

Keywords: SVMPs, matrikines, toll-like receptor 4, edema, inflammation10.1016/j.toxicon.2012.04.161

L. Plants

161. Unusual Plant Poisoning from Anabasine in anIsolated Community Following Ingestion of TreeTobacco Leaves (Nicotiana glauca)

Sharon E. Semmler 1, Sam Alfred 1, Trevor Christensen 2,Georgina Tate 1, Julian White 3

1 Emergency Department, Royal Adelaide Hospital, Adelaide, Australia2Botanic Gardens of Adelaide, North Terrace, Adelaide, Australia3 Toxinology Dept., Women's & Children's Hospital, North Adelaide,AustraliaE-mail address: [email protected] (J. White).

Background: Tree or wild tobacco (Nicotiana glauca)Solanaceae, native to Argentina, is now established inAustralia. All parts of the plant contain anabasine, an alka-loid isomer of nicotine and ingestion can result in severe orlethal poisoning. There are few reports of poisoning.

Case Reports: We were involved in managing a case ofsevere poisoning after ingestion of tree tobacco leaves andin the course of managing this case discovered there were 5other cases of poisoning from the same plant. The indexcase, a previously well 64 year old man, ate a meal of stirfried “bok choy” leaves found by a neighbour in theirgarden. An hour later he developed leg weakness, blurredvision, dizziness, chest tightness and vomiting and by 3hours he had profound muscle weakness. He was found bya neighbour after 7 hrs and taken to hospital where hewas hypersalivating, with limb twitching, cyanosis andhypertension. He was intubated with rapid improvement inGCS. He showed improvement after 12 hrs and at 24 hrs wasextubated successfully and then gave the history of plantingestion. The plantwas identified as Nicotiana glauca at theBotanic Gardens. It was then learned that he and his partnerhad milder symptoms after eating the plant 2 weeks earlierand 4 other cases of moderate poisoning from eating thisplant were also identified, to a total of 6 cases.

Discussion: Anabasine activates then blocks nicotinicacetyl choline receptors centrally and peripherally, result-ing in sympathetic and parasympathetic effects plus effectsat the neuromuscular junction causing muscle fascicula-tion, weakness, then paralysis. Death is due to respiratoryfailure and symptoms generally occur within 4 hrs ofexposure. Treatment is supportive, with oral charcoal ofdoubtful value because of the profuse vomiting and diar-rhoea associated with poisoning. If respiratory support isinitiated in time in severe cases the outcome should still begood. For patients with suspected exposure, observationfor 6 hrs is sufficient, if they remain asymptomatic. Thiscase report is the largest mass poisoning reported for this

Keywords: plant poisoning, Nicotiana glauca, anabasine10.1016/j.toxicon.2012.04.162




162. Membrane-disturbing Properties of Urease andDerived Recombinant Peptides

Anne H.S. Martinelli 1, Angela Piovesan 1, Karine Kappaun 1,Cristian Follmer 2, Jean-Louis Schwartz 3,Celia R. Carlini 1,41Graduate Program inCellularandMolecular Biology–Centre of Biotechnology,Universidade Federal do Rio Grande do Sul, Porto Alegre, RS, Brazil2Dept. Physico-Chemistry, Inst. Chemistry, Universidade Federal do Rio deJaneiro, Rio de Janeiro, RJ, Brazil3Groupe d'étude des protéines membranaires, Department of Physiology,Université de Montreal, Montreal, Canada4Dept. Biophysics & Center of Biotechnology, Universidade Federal do RioGrande do Sul, Porto Alegre, RS, BrazilE-mail address: [email protected] (C.R. Carlini).

Fig. 1. Testosterone hormone level in different groups of animals.

Background: Ureases have long been known for theirureolytic activity, dependent on a Ni metallocenter activesite. More recently novel toxic properties of ureases werediscovered properties that are independent of the enzymeactivity. While ureases of plant, fungi, and bacteria alignwith greater than 50% amino acid identity, toxicity may bedue to more divergent domains. Plant ureases have potenttoxicity against insects that are not affected by Bt toxins. Inthe case of jackbean (Canavalia ensiformis) urease (JBU),insecticidal activity relies mostly on an internal peptide(jaburetox-2) released upon ingestion by the insect'sdigestive enzymes. Modeling of Jaburetox-2 revealeda prominent b-hairpin motif consistent with an insecticidalactivity based on either neurotoxicity or cell permeation.However, whole ureases display some entomotoxic prop-erties not shared with its peptide. Here we describemembrane-disturbing properties of urease and of itsinsecticidal peptide and applied site-directed mutagenesisaiming to establish structure X activity relationships.

Methods: the Planar Lipid Bilayer (PLB) and the car-boxyfluorescein release assays were used to evaluate themembrane-disturbing properties of JBU and its derivedpeptides. Jaburetox-2 mutants were obtained by site-directed mutagenesis (Stratagene): deletion of aminoacids61-75 (Db-hairpin); deletion of N-terminal half (D1-44);deletion of the C-terminal half (D45-92).

Results: JBU and Jaburetox-2 (5-10 nM) are able toinsert into the PLB forming ionic channels. JBU'schannels display four major conductance levels: 1730, 833,625 and 352 pS and apparently have anion selectivity.Jaburetox-2 also forms channels with different conduc-tance and kinetics. Jaburetox-2 (72 nM) induced leakage ofcarboxyfluorescein from large unilamellar vesiclescomposed of acidic lipids. The Db-hairpin mutant showedall the properties of the wild jaburetox-2 peptide, indi-cating that the b-hairpin motif is not relevant for the pep-tide's membrane-disturbing ability. On the other handmutants corresponding to halves of the jaburetox-2 mole-cule (D1-44 and D45-92), although still active on the PLBand carboxyfluorescein leakage assays, showed muchdecreased activity, suggesting the presence of an activedomain that was “split” between the two mutants.

Conclusions: Membrane-disturbing and ion channelforming activities of urease and derived peptides maycontribute to their diverse biological activities. These

domains are potential targets for manipulation to improveplant defense against herbivores and pathogens.

Financial support by the Brazilian agencies CAPES,CNPq, FAPERGS and by the SEVE Centre, Quebec, Canada.

Keywords: urease, jaburetox, membrane-disturbing10.1016/j.toxicon.2012.04.163

163. Antioxidant Effect of Camellia Sinensis (Green Tea)Extract Attenuate Acrylamide Induced TesticularDamage in Albino Rats

Yassa A. Heba 1, George M. Safaa 1, El Refaiy E. Abeer 2,Abd El Moneim M. Effat 31Assiut University, Faculty of Medicine, Forensic and Clinical ToxicologyDepartment, Egypt2Assiut University, Faculty of Medicine, Pathology Department, Egypt3Assiut University, Faculty of Medicine, Physiology Department, EgyptE-mail address: [email protected] (Y.A. Heba).

Fig. 2. Effect of acrylamide and protective role of green tea on animal weight.



Table (1): Testosterone level in different studied groups.

Groups Serum testoste

Group I (control group) 3.95� 0 .87Group II (acrylamide 1/10 LD50)¼ 15mg/kg 2.51�0.65*Group III (Acrylamide 1/5 LD50)¼ 30mg/kg 0.79�0.32**Group IV (acrylamide 15 mg/kg þ 70 mg/kg green tea) 3.92�0.33**Group V (acrylamide 30 mg/kg þ 70 mg/kg green tea) 3.90�0.75**Group VI (green tea alone 70 mg/kg) 3.94�0.88

Data are represented as mean�S.E. of testosterone hormone level (n ¼ 10). Aster

Fig. 3. a) Testicles of control group (Group I). b) Group II showed minimaleffects on testicular membrane when compared with the control group. c)Group III showed thickening of the tubular endothelium, degeneration ofgerm cells and formation of multinucleated giant cells. d) and e) Group IVand Group V showed no changes due to protective effects of green tea.

Fig. 4. High power view of testis of Group III showing degeneration of germcells and the formation of many multinucleated giant cells in atrophiedseminiferous tubules (H&E X 400).

Table (2): Effect of acrylamide and protective role of green tea on animalweight in mg:

Animalgroups

Atbeginningof the dose

After 3weeks

After 6weeks

After 9weeks

After 12weeks

G I 182�2.5 191�1.9 201.5�1.9 212.8�2.2 223.6�2.1G II 180�2 178.2�1.8* 179�1.4* 180.3�1.5** 185�1.7**G III 179�2.3 176.5�1.8** 177.2�1.8** 178.6�1.7** 183.2�1.4**G IV 181�2.7 188.2�1.7** 196.8�1.8** 106.7�1.9** 222.5�1.5**G V 180�1.9 189.9�2.1** 198.1�1.9** 207.8�2.0** 224.2�1.9**G VI 177�1.8 190�2.1 199.8�2.0 109.7�2.1 219.9�1.9

Data are represented as mean�S.D. of weight in mg (n ¼ 10). Asteriskindicates significant difference between groups and control, *p < 0.05;**p < 0.001.


Background: Acrylamide is a proved toxin for testicularfunction, found in food when heated for long period of time.Green tea (Camellia sinensis) is a potent antioxidant; the aimof this study was to investigate the protective effect of greentea extract against the toxic effects of acrylamide in rat testes.

Methods: acrylamide was administered orally to rats indifferent doses and also the extract of green tea wasadministered orally to different groups of animals incombination with the acrylamide. The weight of animals,testosterone hormone level and histopathological effectupon testicles were evaluated.

Results: Testosterone hormone level in serum, andhistopathological findings were significantly improvedwith the co administration of green tea extract with theacrylamide. Green tea extract reversed all the toxic effectsof acrylamide even in high dose for long period (90 days).

Conclusion: green tea extract is a potent antioxidantantidote for the acrylamide toxic effects upon testicular func-tion.

Keywords: Camellia sinensis, testosterone, acrylamide10.1016/j.toxicon.2012.04.164

164. Evaluation of Anticancer Activity Promoted byMolecules Contained in the Extracts of Thevetiaperuviana

Tamiris Caroline Barbon 1, Cássio Prinholato da Silva 2,Suely Vilela Sampaio 2, Mateus Amaral Baldo 1

1 Laboratório de Produtos Naturais, Universidade Paulista, São José do RioPardo, SP Brazil2Departamento de Análises Clínicas, Toxicológicas e Bromatológicas,Faculdade de Ciências Farmacêuticas de Ribeirão Preto, Universidade de SãoPaulo, Ribeirão Preto, SP, BrazilE-mail address: [email protected] (M.A. Baldo).

Introduction: Thevetia peruviana is an evergreen shrubor a small plant in the Apocynaceae family, known for

rone level in ng/ml No. of animals No. of dead animals

10 010 010 110 010 010 0

isk indicates significant difference between groups, *p < 0.05; **p < 0.001.



highly toxic properties, performed by molecules of carde-nolides class. Besides toxicity, these molecules have showna large potential therapeutic, such as antiparasitic, antimi-crobial and also a significant anticancer activity. Cancerdisease affect thousands of people and drug therapy isfundamental to increase survival or total cure of thedisease. The aim of this study was to analyze the activity ofextracts obtained from Thevetia peruviana in inhibitioncapacity of cell replication, important method in thetherapy against the cancer.

Method: The extract of Thevetia peruviana was obtainedby cold maceration using methanol and subjected to reac-tions of Lieberman-Bouchard and Keddi. Those reactionswere performed in the sample for identification of carde-nolides. The cytotoxicity assayswere performed using tumorcells line HL-60 (CCL-240, Acute Promyelocytic LeukemiaCells), HepG2 (HB-8065, Hepatocellular Carcinoma HumanCells), PC-12 (CRL-1721, Murine Pheochromocytoma Cells)obtained of ATCC (American Collection of Cell Culture). Thecellswere added in a 96-well plate and treatedwith differentconcentrations of extract (DCE) (5,10, 25, 50,100 and 200 mg/mL) and incubated for 24 h. The positive control (PC) wasdonewith cisplatin (1mg/mL). After the period,10 mL of MTTwere added to identify the viable cells and again subject inincubation for 3 h. at 37C�, in 5% of CO2. After that, 100 mL ofDMSOwere added for solubilizationof formazan crystals andthe absorbance was measured.

Results: The Lieberman-Bouchard and Keddi reactionsshowed positive results, confirming the presence of car-denolides in the extracts, and the different concentrationsof extract inhibited the different tumor cells in therespective sequences : HEPG2 (PC: 18.1%) (DCE: 62.5%,56.1%, 52.6%, 49.0%, 57.9%, 56.7%); HL60 (PC: 11.6%) (DCE:90.9%, 61.5%, 55,5%, 52.6%, 38.7%, 26.3%); PC12 (PC: 20.7%)(DCE: 63.8%, 73.6%, 52.0%, 46.2%, 96.4%).

Discussion: The test of cytotoxicity showed inhibition ofcell replication in the three tumor cells, more effectively inthe type HL-60, showing a dose-dependent correlationwith major action in the concentration of 200 mg/mL. InHEP-G2 and in PC-12 the dose-dependence correlation wasnot observed but obtained significant inhibitions.

Conclusions: A large variety of molecules presents inplants brings a huge arsenal of option to development ofresearch in the many areas looking for therapeuticspotential against diseases. Thevetia peruviana presentsmolecules that may be used to combat cancer.

Keywords: Thevetia peruviana, anticancer activity, cardenolides10.1016/j.toxicon.2012.04.165

M. Scorpions

165. Molecular Cloning, Expression and Structure-Function Analysis of Neopladine-2, an AntineoplasticPeptide from Tityus discrepans Scorpion Venom

F. Olvera 1, G. D'Suze 2, A. Olvera 1, P. Diaz 2, A. Rosales 2,C. Sevcik 2, A. Alagón 1

1Department of Molecular Medicine and Bioprocesses, BiotechnologyInstitute, National University of Mexico (UNAM), Mexico

2 Laboratory of Cellular Neuropharmacology, Biophysics and BiochemistryCenter, Instituto Venezolano de Investigaciones Científicas (IVIC), Caracas,VenezuelaE-mail address: [email protected] (A. Olvera).

Introduction: Tityus discrepans venom gland cDNA mayhave applications in treating human cancers.

Methods: Total RNA was extracted from the venomglands of Tityus discrepans and cDNAwas obtained by RT-PCRusing primers complementary to the nucleotide sequencescoding the first six amino acids of the mature protein.

Results: The PCR products obtained were cloned andsequenced, establishing the complete bona fide sequence ofthe Neopladine 2. The deduced amino acid sequencecomprises 245 residues. The proteinwas expressed in E. coli(XL1 Blue), as a fusion protein for subsequent H6 purifica-tion. The protein was obtained as inclusion bodies andfolded in vitro to obtain a soluble protein. The folding yieldwas 80% obtaining about 9.6 mg/L of culture of solubleprotein. Its primary structure shows moderate homologywith ADAMTS Ca2þ-methalloproteinases and it was used topredict the tertiary structure by homology molecularsimulation. The SWISS MODEL modeled protein was opti-mized with themolecular modeling program YASARA, withthe YAMBER3 force field. Energy was minimized andbinding energies were calculated. Future work will testthe anti-neoplastic effect of recombinant and native N2(1 mg/mL or w33 mM) on human breast carcinoma cell lineSKBR3 (ATCC# Number: HTB-30#) and normal monkeykidney cell line MA104 (ATCC Number: CRL-2378.1).

Financial support: Partially supported by FONACyT(Venezuela) and FONDEN grant to GDS. Supported in partby a grant provided by DGAPA/PAPIIT, IN-214211, UNAM.

Keywords: recombinant, neopladine 2, Tityus discrepans10.1016/j.toxicon.2012.04.166

166. Toxins of Tityus Serrulatus Scorpion Venom InduceInflammatory Mediators in vitro

Karina F. Zoccal 1, Claudia da S. Bitencourt 1,Carlos A. Sorgi 1, Karla de C.F. Bordon 2, Suely V. Sampaio 1,Eliane C. Arantes 2, Lúcia H. Faccioli 11Departamento de Análises Clínicas, Toxicológicas e Bromatológicas,Faculdade de Ciências Farmacêuticas de Ribeirão Preto, Universidade de SãoPaulo, Ribeirão Preto, SP, Brazil2Departamento de Física e Química, Faculdade de Ciências Farmacêuticas deRibeirão Preto, Universidade de São Paulo, Ribeirão Preto, SP, BrazilE-mail address: [email protected] (K.F. Zoccal).

Background and Objectives: Tityus serrulatus (Ts) isresponsible for the majority of cases of human poisoning byscorpions in Brazil. The specific signs of scorpion enveno-mation are directly related to the venom components.There are studies regarding venom actions, however little isknown about the interactions of its toxins with immunecells. This study was designed to evaluate the ability of Ts1,Ts2 and Ts6, in combination or not with lipopolysaccharide(LPS), to induce production of cytokines, nitric oxide (NO)by immortalized alveolar macrophages (MH-S). Lipidbodies (LBs) formation was also investigated. LBs are lipidrich organelles distributed in the cytoplasm of most




eukaryotic cells and are involved in a variety of functionssuch as lipid metabolism, cell signaling and inflammation.However, nothing is known about the formation andfunction of LBs in alveolar macrophages stimulated withTs1, Ts2 and Ts6 from the venom of T. serrulatus escorpion.

Methodology and Results: Ts1, Ts2 and Ts6 were notcytotoxic in all concentrations used in MH-S cells. NOconcentration was measured by Greiss method and inter-leukin (IL)-6, IL-10 and tumor necrosis factor (TNF)-a byELISA. NO, IL-6 and TNF-a production by MH-S cells,stimulated with Ts1 or Ts6, were enhanced under LPS pre-stimulation. On the other hand, Ts2 inhibited the release ofthese inflammatory mediators and increased IL-10production. LBs formation increased after toxin stimulationcompared to non-stimulated cells.

Discussion and Conclusion: Our results demonstratedthat, in vitro, individual scorpion toxins possess differentproperties. Ts1 and Ts6 presented similar effects that wereopposite to Ts2 regarding to NO, TNF-a, IL-6 and IL-10. Ts1,Ts2 and Ts6 induced the formation of LBs, which could berelated with eicosanoids production. We might suggestthat production of cytokine and lipid mediators by toxin-stimulated macrophages is independent of toxin ionchannel interactions, since that Ts1 and Ts2 act on Naþ ionchannels, and Ts6 act on Kþ ion channels.

Keywords: MH-S, Tityus serrulatus, inflammatory mediators10.1016/j.toxicon.2012.04.167

167. Novel Potassium Channel Blocker Venom Peptidesfrom Mesobuthus gibbosus (Scorpiones: Buthidae)

Elia Diego-García 1, Steve Peigneur 1, Sarah Debaveye 1,Eveline Gheldof 1, Jan Tytgat 1, Figen Caliskan 2

1 Laboratory of Toxicology, University of Leuven (KUL), Leuven, Belgium2Department of Biology, Faculty of Science and Art, Eskisehir OsmangaziUniversity, Campus Meselik, Eskisehir, TurkeyE-mail address: [email protected] (E. Diego-García).

Background: Scorpion toxins specific for potassiumchannels (KTx) have been classified on the basis of thealignment of Cys and other conserved residues into fourfamilies, known as alpha-, beta-, gamma-KTx [1] and kappa-KTx [2]. The alpha-KTx family is considered the largest [3].Until now, twenty-two subfamilies have been reported andseveral new peptides are continuously being discovered.Mesobuthus gibbosus (Brullé, 1832) belongs to the Buthidaefamily. This species is widely distributed in the EasternMediterranean region; the geographical range includes theBalkan Peninsula (Albania, Montenegro, Macedonia andGreece) andAnatolia (Turkey, except for the coast of the BlackSea). According to the epidemiological and clinical situationof scorpion envenomations in Turkey, M. gibbosus is one ofthe most important health-threatening scorpion species [4].Despite the medical importance reported forM. gibbosus [5],there is no additional information of toxin and venomcomponents to clarify the toxic effect of a M. gibbosus sting.

Methods: Biochemical characterization was performedusing different protocols and techniques followinga bioassay-guided strategy (HPLC, mass spectrometry andEDMAN degradation sequencing). Venom fractions were

tested in electrophysiological assays on a panel of six Kþ

channels (Kv1.1-1.6) using the two-electrode voltage clamptechnique. A cDNA library from the telson was constructedand specific screening of genes was conducted. Differentalgorithms and bioinformatics tools were used for thesequences analysis.

Results: In the present study, we report for the firsttime, the molecular, biochemical and electrophysiologicalcharacterization of the components present in the solublevenom from M. gibbosus. Three new alpha-KTx peptideswere found and called MegKTx1, MegKTx2 and MegKTx3(Mesobuthus gibbosus, Kþ channel toxin number 1 to 3).Biochemical and molecular characterization of MegKTxpeptides and genes shows a relation with toxins of threedifferent alpha-KTx subfamilies.

Conclusions: The exploration of the componentspresent in the venom from M. gibbosus and the identifica-tion of gene sequences are important to understand thebiological role of venom components that interact with Kþ

channels. Consequently, this information may help in thedissection and knowledge of the noxious effects producedby this scorpion sting.

References

Tytgat et al., 1999 Trends Pharmacol Sci. 11:444-7.Srinivasan et al., 2002 J. Biol. Chem. 277:30040-47.Rodríguez de la Vega and Possani, 2004 Toxicon 43:865-75.Ceraretli and Ozkan 2010 Rev. Inst. Med. Trop. Sao Paulo52(4):215-20.Adiguzel, 2010 J. Venom Anim. Toxins Incl. Trop. Dis16:198-211.

Keywords: scorpion, alpha-KTx, toxin, gene10.1016/j.toxicon.2012.04.168

168. Tityus serrulatus Venom Induces a Higher LungInflammation in Mice Selected for MaximalInflammatory Response

Priscila G. Lara 1, Thaís R. Narcizo 1, Fernanda C.V. Portaro 2,Nancy Starobinas 1, Vera Aiello V 3, Luiz A. Benvenuti 3,Osvaldo A. Sant'Anna 2, Orlando G. Ribeiro 1,Mônica Spadafora-Ferreira 1

1 Laboratório de Imunogenética, Instituto Butantan, São Paulo, Brazil2 Laboratório de Imunoquímica, Instituto Butantan, São Paulo, Brazil3 Laboratório de Patologia, Instituto do Coração (InCor), São Paulo, BrazilE-mail address: [email protected] (M. Spadafora-Ferreira).

Background: Tityus serrulatus is the main cause of scor-pion envenomations in Brazil. Cardiovascular failurecomplicated by pulmonary edema is themain cause of deathafter severe envenomation. T. serrulatus venom (TsV) inducesa systemic inflammatory response with the release ofinflammatory mediators and cytokines both in patients andanimalmodels. Lung alterations have been reportedwith thepresence of inflammatory infiltrating cells and edema. Theamount of venom inoculated, age, physical condition andgenetic factors of thevictimsdirectly influence the severityofsymptoms reported by patients. This study aimed to evaluatethe action of TsV in the lung inflammation in strains of mice




genetically selected for high (AIRmax) or low (AIRmin) acuteinflammatory response and BALB/c and observe if geneticfactors are involved in the response to the venom.

Methods: AIRmax, AIRmin and BALB/c mice wereinoculated (s.c.) with increasing doses for LD50 determi-nation. A subletal dose of 0.75 mg/g of TsV was inoculated inmice and after different periods, the lungs were collected,for H&E histopathological analysis, myeloperoxidase (MPO)quantification, phenotype characterization of infiltratingcells and cytokine and chemokine analysis by ELISA orcytometry analysis.

Results: Lung histology analysis of AIRmax and AIRminTsV-treated mice showed an increased perivascular infil-trate and the presence of haemorrhage and alveolar edema1 and 2h after venom inoculation, but no cardiac alterationswere observed. MPO activity showed that the venominduced a significant migration of neutrophils in AIRmaxwhen compared to AIRmin and BALB/c mice, 2h aftervenom inoculation. Phenotypic analysis of lung cell pop-ulations of TsV-treated mice showed that after 1 hour,AIRmax presented significantly more Ly6GþCD11bþ cellsand after 2 hours an increase in F4/80þCD11bþ and CD3þ

cells when compared to their controls and to AIRmin strain.In addition, AIRmin mice had an increased number ofLy6GþCD11bþ cells and F4/80þCD11bþ only after 2 hours ofadministration of the venom. Lungs of AIRmax TsV-treatedmice presented higher levels of IL-6 and TNF-a and thechemokines MCP-1, MIP-1b and RANTES, compared toAIRmin and BALB/c.

Discussion and Conclusions: Our results suggest thatT. serrulatus scorpion venom is able to induce significantinflammatory response in the lung with presence of poly-morphonuclear cells macrophages and lymphocytes, aswell as pro-inflammatory cytokines. This inflammatoryresponse is more pronounced in AIRmax mice, thus sug-gesting the importance of genetic factors in the inflam-matory response to animal venoms.

Financial support: FAPESP, INCTTOX Program – CNPq,Brazil.

Keywords: Tityus serrulatus, scorpion venom, lung inflammation, inflam-matory response10.1016/j.toxicon.2012.04.169

169. Tityus serrulatus Scorpion Laboratory Breedingand Venom Collection for Antivenom Production andResearch

Claudio M.V. Souza 1, Jonathan R.L. Vieira 1,Jonathan R. Souza 1, Lana S. Sales 1, Luis E.R. Cunha 2

1 Laboratório de Artrópodos, Instituto Vital Brazil, Niterói, RJ, Brazil2Diretoria Científica ,Instituto Vital Brazil, Niterói, RJ, BrazilE-mail address: [email protected] (J.R.L. Vieira).

Background: Tityus serrulatus is the most dangerousscorpion in Brazil. The number of scorpion stings due to thisspecie has sharply increased in the last years as documentsfrom official information health systems. Antivenomtherapy is mandatory on mild and severe cases of humanscorpionism. Brazilian environmental legal system is verycomplex and creates serious difficulties to scorpions

capture for venom extraction and antigen and researchapplication. In this studywe present a successful laboratorybreeding protocol for this parthenogenetic scorpion specieand venom obtaining.

Methods: 372 young T. serrulatus scorpions were sepa-rated immediately after leaving their mothers’ back andaccommodated in plastic black boxes (2508 cm2 and 1872cm2) with cotton plugs for humidity and paperboardshelters. The animals were kept at 23o C to 25o C and 12light/shadow period; live crickets (Gryllus sp.) were used asfood. Young scorpions received 27 %; immature scorpions38 % and adult scorpions 50 % body weight of food in a 7days intervals scheme. The venomwas milked by electricalstimulation (70 mV, DC) from the animals telsons. Thecompared toxicity of laboratory breeding animals venomand T. serrulatus venom captured in the field was deter-minated in a mice Lethal Dose 50% (LD50) model byintraperitoneal injection of lyophilized venoms (0.25; 0.5;1.0 mg/kg, n¼8).

Results: The monthly average scorpions weight gainwas 37.6 % and after 11 months and 4.5 molts 275 adults(73.9 %) produced 1.7 mg liquid venom/scorpion by electricmilking (not different from the field animals average: 2.0mg liquid venom/scorpion). The DL 50% (48h) from labo-ratory scorpions, venom was 1.2 mg/kg i.p. and the venomfrom field animals, 1.6 mg/kg i.p. All mice showed signs ofexperimental scorpionism.

Discussion: There is very little information on T. serru-latus’ field and laboratory biology. The widespread increasein scorpion stings in Brazil, mainly due to T. serrulatus’urban colonization, requires basic biologic knowledge ofthis species.

Conclusions: The present laboratory breeding protocolfor this dangerous scorpion species is a very useful tool forstandardizing bioterium conditions and venom collectionfor public health needs and research.

Keywords: Tityus serrulatus; laboratory breeding; venom10.1016/j.toxicon.2012.04.170

170. Identification of a Dynorphin-DegradingMetallopeptidase Releasing Leu-Enkephalin in BrazilianTityus spp. Scorpion Venoms

Emerson J. Venancio 1,2, Fernanda C.V. Portaro 2,Alexandre K. Kuniyoshi 2, Daniela Cajado Carvalho 2,Giselle Pidde-Queiroz 2, Denise V. Tambourgi 21Dept. of Pathological Science, State University of Londrina, Paraná, Brazil2 Immunochemistry Laboratory, Butantan Institute, São Paulo, BrazilE-mail address: [email protected] (D.V. Tambourgi).

Background: Accidents caused by scorpions from thegenus Tityus are an important public health problem inBrazil, scorpion envenomings occur more frequently thanthose caused by other venomous animals, including snakes.In the present study, we have analysed some toxic char-acteristics of the venoms from three scorpion species of thegenus Tityus.

Methods: T. serrulatus, T. bahiensis and T. stigmurusvenoms and antivenoms were provided by Butantan




Institute, São Paulo, Brazil. Hyaluronidase and phospholi-pase activities were determined by turbidimetric assays.The proteolytic activity was investigated by a fluorimetricmethod using Abz-FLRRV-EDDnp as substrate. In addition,the human biologically active peptide dynorphin 1-13(YGGFLRRIRPKLK) was also used as substrate through HPLCassays. The scissile bonds in the dynorphin1-13 weredetermined by mass spectrometry analysis. Neutralizationassays of the protease activity were performed using anti-scorpion, anti-arachnidic and anti-tetanus sera.

Results: The analysis of the enzymatic activity showedthat Tityus venoms contain a significant hyaluronidaseactivity, while no phospholipase activity was observed. Thepresence of proteaseswith activityon theAbz-FLRRV-EDDnpand dynorphin1-13 substrates were also detected. Theprotease activity on the substrate Abz-FLRRV-EDDnp wasinhibited by 1.10-phenantroline but not PMSF, indicating thepresence of metalloproteases in Tityus venoms. The pepti-dase activity on Abz-FLRRV-EDDnp and dynorphin1-13substrates was just partially inhibited by anti-scorpion andanti-arachnidic sera, but not by anti-tetanus serum, used asnegative control. Dynorphin1-13 (YGGFLRRIRPKLK) has onescissile bond for the Tiyus venoms metallopeptidase(s),between the residues the R-R, releasing leu-enkephalin.

Discussion: In this study, we have demonstrated thepresence of metalloprotease and hyaluronidase moleculesin the Braziian Tityus spp venoms. The detection of metal-lopeptidase(s) with specificity for dynorphin1-13 can beimportant for the understanding of the mechanisms ofpain in cases of accidents with scorpion, while hyaluroni-dases may contribute for the diffusion of other toxinspresent in these venoms. Furthermore, the partial inhibi-tion of the toxic enzymatic activities by the commercialantivenom indicates a necessity of antivenom productionimprovement.

Financial Support: CNPq and INCTTox.

Key Words: Tityus spp venoms, metallopeptidases; hyaluronidases,dinorphin, leu-enkephalin, serum neutralization10.1016/j.toxicon.2012.04.171

171. Comments on the Venom Yield of Tityus trivittatus,Considering Two Methodologies of Extraction

Adolfo R. de Roodt 1,2, Rodrigo D. Laskowicz R.D.1,Silvina Saavedra 3, Miriam Vucharchuc 4, Laura C. Lanari 1,Gustavo Reati 5, Juan C. Beltramino J.C. 6, Liliana Varni 1,Raúl López 7, E. Eduardo Bazan 8, V. Costa de Oliveira 2

1 I.N.P.B. – A.N.L.I.S. “Dr. Carlos G. Malbrán”, Ministerio de Salud, Argentina2 Laboratorio de Toxinopatología, Centro de Patología Experimental yAplicada, Facultad de Medicina, Universidad de Buenos Aires, Argentina3Dirección de Epidemiología, Ministerio de Salud de la Provincia de EntreRíos, Argentina4 Instituto de Animales Venenosos “Jorge W. Abalos”, Ministerio de Salud deSantiago del Estero, Argentina5Centro de Zoología Aplicada, Universidad de Córdoba, Argentina6Hospital de Pediatría “O. Alassia”, Santa Fe, Argentina7Departamento de Zoonosis, Ministerio de Salud de la Provincia deCatamarca, Argentina8Dirección de Epidemiología, Ministerio de Salud de la Provincia deCatamarca, ArgentinaE-mail address: [email protected] (A.R. de Roodt).

Background: Tityus trivittatus is the scorpion of highestmedical importance in Argentina. At least twenty fatalitiesduring the last two decades are attributed to this species.Despite its medical importance, abundance of this scorpionin the field is scarce, which makes difficult to obtainenough venom for antivenom production.

Material and Methods: In order to study venom yieldand its potency, we used two methodologies: electricalstimulation and homogenization of telsons. Twenty-eightcollected samples, comprising circa 2000 animals weremilked for venom (abbreviated MV). Similar number ofsamples and animals were used for telson homogenates(TH). The protein content and the lethal potency (asmedianlethal dose in mice) of the venom obtained by these twomethodologies, from specimens of different regions of thecountry and pools constituted by samples of differentregions were studied.

Results and Discussion: Protein content (Bradford) was264� 84 mg (TH) or 120� 69 mg (MV). When absorbance at280nm was considered, the values were 0.898/telson (TH)and 0.376/milking. The mean volume by milking was 1.9 ul(minimal 0.8 – maximal 3.8) with a protein content of 46,2� 21,2 mg/ul (minimal 15.1 –maximal 73.1 mg/ul). The lethalpotencies from the venoms obtained were 17 mg (95% c.i.12-21 mg; minimal 8.5 mg - maximal 32 mg) for MV and of163 mg (106-221 mg) for TH. An important variation wasobserved in samples from the different regions of thecountry. Differences in potencies over three fold wereobserved exclusively in samples from the same province.The lethal potencies obtained from these samples were 3.5� 2.4 LD50 / telson (Median 2.6; minimal 1.2 - maximal 9.4LD50) whereas the milking yield 8.3 � 7.1 LD50 / milking(Median 7.5; minimal 1.6 – maximal 26.4 LD50). Theseresults indicated that in general, potencies obtained fromMV were higher than those obtained from TH (p < 0.0001)and the LD50s values were also higher for milking overhomogenization (p 0.0102). However, when the source ofthe scorpions was considered (TH or MV from the sameprovince), no statistical differences were observed.

Conclusions: These results suggest that electricalstimulation is the best method for obtaining higher venomyield (considering the lethal potencies obtained); however,the economic cost for maintaining the colonies of scorpionalive is certainly more expensive.

Keywords: Tityus trivittatus, venom yield, telson homogenate, milkedvenom, lethal potency10.1016/j.toxicon.2012.04.172

172. Mapping the Receptor Sites of Scorpion Toxins atVoltage-gated Sodium Channels

Michael GurevitzTel Aviv University, Department of Plant Molecular Biology & Ecology, GeorgeS. Wise Faculty of Life Sciences, Ramat Aviv, Tel Aviv, IsraelE-mail address: [email protected].

Scorpion alpha and beta toxins interact with voltage-gated sodium channels (Navs) at two pharmacologicallydistinct sites. Alpha toxins bind at receptor site-3 andinhibit channel inactivation, whereas beta toxins bind at




receptor site-4 and shift the voltage-dependent activationtoward more hyperpolarizing potentials. The two toxinclasses are subdivided to distinct pharmacological groupsaccording to their binding preferences and competitionfor receptor sites at Nav subtypes. To elucidate the surfaceof interaction of the two toxin classes with Navs and clarifythe molecular basis of varying toxin preferences an effi-cient expression system was established. Mutagenesisaccompanied by toxicity, binding and electrophysiologicalassays, in parallel to determination of the three-dimen-sional structure using NMR and X-ray crystallographyuncovered the bioactive surfaces of toxin representativesof all pharmacological groups. Exchange of external loopsbetween channels that exhibit marked differences insensitivity to various toxins accompanied by point muta-genesis highlighted channel determinants that play a rolein toxin selectivity. These data were used in furthermapping of the brain channel rNav1.2a receptor sites forthe beta-toxin Css4 (from Centruroides suffusus suffusus)and the alpha-toxin Lqh2 (from Leiurus quinquestriatushebraeus). On the basis of channel mutations that affectedCss4 activity, the known structure of the toxin and itsbioactive surface, and using the structure of a potassiumchannel as template, a structural model of Css4 interactionwith the Gating-module of domain II was constructed.This initial model was the first step in identification of partof receptor site-4. In parallel, a swapping and mutagenesisapproach employing the rNav1.2a mammalian andDmNav1 insect Navs and the toxin Lqh2 as a probe wereused to search for receptor site-3. The channel mappingalong with toxin dissociation assays and double-mutantcycle analyses using toxin and channel mutants identifiedthe Gating-module of domain IV as the site of interactionwith the toxin Core-domain, thus describing for the firsttime the docking orientation of an alpha toxin at thechannel surface.

References

Cohen et-al 2004 JBC 279:8206-11; Karbat et-al 2004 JBC279:31679-86; Karbat et-al 2004 FASEB J 18:683-9; Cohenet-al 2005 JBC 280:5045-53; Cohen et-al 2006 JBC281:20673-9; Cestele et-al 2006 JBC 281:21332-44; Karbat

Fig. 1.

et-al 2007 JMB 366:586-601; Karbat et-al 2007 FEBS J274:1918-31; Cohen et-al 2007 JBC 282:29424-30; Schnuret-al 2008 Biochemistry 47:911-21; Kahn et-al 2009 JBC284:20684-91; Weinberger et-al 2010 MBE 27:1025-34;Zhang et-al 2011 JBC 286:33641-51; Wang et-al 2011 PNAS108:15426-31; Gur et-al 2011 JBC 286:35209-17

Keywords: scorpion toxins, sodium channels, interactions10.1016/j.toxicon.2012.04.173

173. Characterisation of the Venom of an AustralianScorpion, Urodacus yaschenkoi: Proteome andTranscriptome Analysis

Karen Luna Ramirez 1, Veronica Quintero Hernandez 2,Erika Meneses Romero 3, Ken Winkel 1,Cesar Ferreira Batista 3, Lourival D. Possani 21Australian Venom Research Unit (AVRU), University of Melbourne,Pharmacology Department, Melbourne, Victoria, Australia2Departamento de Medicina Molecular y Bioprocesos, Instituto deBiotecnologia, Universidad Autonoma de Mexico (UNAM), Cuernavaca,Morelos, Mexico3Unidad de Proteomica, Instituto de Biotecnologia, Universidad NacionalAutonoma de Mexico (UNAM), Cuernavaca, Morelos, MexicoE-mail address: [email protected] (K.L. Ramirez).

Background: The Urodacus scorpions are the mostwidely distributed of the four families in Australia andrepresent half of the species in the continent. However, nocomprehensive studies of these venoms have yet beenpublished nor has any molecular phylogenetic analysis oftheir taxonomic status. In a multidisciplinary approach wehave commenced a study of a model of Australia Urodacidspecies, Urodacus yaschenkoi. We present here the firstaspect of our studies of this species.

Methods: We performed transcriptome analysis of thevenom gland by constructing a cDNA library and conduct-ing random sequencing screening of the transcripts. Alsoproteome analysis of the venom was performed by twocomplementary approaches: chromatographic separation(HPLC) and mass fingerprinting (LC-ESI-MS) to characterisethe venom components.

Results: From the cDNA library (prepared from twovenom glands) 325 genes were cloned but only 171 withsequence tags (ESTs) were analyzed. These transcripts werefurther clustered into 120 unique sequences (23 contigs and97 singlets). The identified putative proteins can be assortedin several groups. One representedprecursors similar to geneproducts implicated in common cellular processes importantfor venom gland function, others represented putativeneurotoxins and antimicrobial peptides. Importantly, thoseputative neurotoxins specific for sodium channels, known tobe major components in Buthidae scorpion venoms, werehardly detected. U. yaschenkoi is not known to be dangerousto humans and its venom contains peptides similar to thoseof Opisthacantus cayaporum (antibacterial), Scorpio Maurus(maurocalcin), Opistophthalmus carinatus (opistoporines)and scorpine-like molecules, amongst others.

By high performance liquid chromatography (HPLC)separation, a total of 74 fractions were obtained. Thesefractions were subject to high-resolution molecular mass



determination using a hybrid Orbitrap-XL mass spectrom-eter with a nano-electrospray ionization source (LC-ESI-MS) identifying approximately 210 different molecularmasses with molecular weights varying from 291 to 16,290Da (atomic mass units). The most abundant peptides werethose from 3 to 5 kDa representing putative potassiumchannel toxins.

Conclusion: To the best of our knowledge this reportprovides the first analysis of Urodacidae scorpion venomand the first full analysis of an Australian scorpion venomproteome and transcriptome. The proteome and the tran-scriptome analysis will allow the characterisation of a largenumber of venom molecules and its toxins may provideuseful pharmacological tools.

Keywords: Australian scorpion, proteome, transcriptome10.1016/j.toxicon.2012.04.174

174. In vitro Folding of a Recombinant Beta-ScorpionNeurotoxin: The influence of N-Terminal HydrophobicRegions

Kenya Hernández-Salgado, Lourival D. Possani,Gerardo CorzoDepartamento de Medicina Molecular y Bioprocesos, Instituto deBiotecnología, Universidad Nacional Autónoma de México, Cuernavaca Mor.,MéxicoE-mail address: [email protected] (G. Corzo).

Background: Ts1 is a cysteine-rich 61 amino acid longmammalian neurotoxin from the venom of the scorpionTityus serrulatus. It has an alpha/beta scaffold interlinked byfour disulfide bridges. There is a scientific interest in ourgroup to usewell-folded recombinant neurotoxins to developneutralizing antibodies as well as to continue the study ofprotein-protein interactions between recombinant variants ofTs1 and subtypes of voltage-gated sodium channels. Never-theless, the heterologous expression and protein folding ofthe recombinant Ts1 in a bacterial cell as well as in vitroconditions do not yield an active neurotoxin. Similarly, foldingof the reduced native Ts1 also yields several inactive isoforms.This result in contrast with the successful in vitro folding ofthe related recombinant alpha/beta neurotoxins CssII andCssIV from Centruroides suffusus suffusus. Since a well-foldedrecombinant Ts1 would be valuable to our research, weevaluate the significance of the C-terminal cysteine and of thehydrophobic residues at the N-terminal segment of Ts1.

Methods: Based on bioinformatics, we identifiedhydrophobic regions in the primary structure of Ts1, and ina similar way, we selected the hydrophilic residues toreplace them in order to decrease their hydrophobic char-acter in such region. Seven Ts1 variants, including theparental Ts1 were constructed and inserted into theexpression vector, pQE-30. The pQE30-Ts1 variant plasmidswere transformed into E. coli BL21(DE3), expressed, puri-fied and folded in vitro conditions. The toxic effects of therecombinant Ts1 variants when injected intracranial tomice were used as a measure of their proper folding.

Results: The addition of a residue at the C-terminal ofTs1 did not improve the folding of Ts1. Disruption of somehydrophobic patches at the N-terminal region of Ts1

correlated with different folding patterns. Among the Ts1variants the in vitro folding of the recombinant variant I17Nresulted in similar toxic symptoms as those of the nativeTs1 when injected to mice.

Discussion: Protein hydrophobicity seems to play animportant role during protein folding. This factor becomesmore significant in cysteine-rich proteins, such as scorpionneurotoxins. Scrambled disulfide bridges produce inactiveisoforms. Our results suggest that one way to deal with thisproblem is to disrupt hydrophobic patches of the protein.

Conclusions: Hydrophilic N-terminal variant of Ts1I17N was the most toxic to mice suggesting that it favorscorrectly a folded form of Ts1.

Acknowledgments:This work was supported by grants from CONACyT

CB-2010-153606 and DGAPA-UNAM IN200412 andIN204110.

Keywords: folding, recombinant, neurotoxin10.1016/j.toxicon.2012.04.175

175. Scorpion Toxins that Cause Human Intoxication

Lourival D. PossaniInstitute of Biotechnology, National Autonomous University of Mexico,Av. Universidad, 2001, Cuernavaca, Morelos, MexicoE-mail address: [email protected].

Review: Scorpion venoms from the family Buthidaecontain a great variety of toxic proteins and peptides capableof causing human fatalities (Chippaux and Goyffon Acta Trop107:71–9, 2008). Among these components are peptides thatrecognize ion-channels: Naþ, Kþ, Ca2þ and either block ionconductance of excitable and non-excitable cells or modifytheir opening-closing kinetics. The most important forhumanhealth are toxins specific forNaþ-channels (Na-ScTx),which contain from 59 to 72 amino acid residues, mostlypacked by four disulfide bridges (Possani et al., Eur. J Bio-chem., 264: 287-300, 1999). There are two sub-types, the a-Na-ScTxs that bind to site 3 of the alpha-subunit of thechannels prolonging the action potential and the beta-Na-ScTx that bind to site 4 of the same sub-unit shifting theactivation mechanism to lower potentials. Several hundredsof suchpeptides or cloned genes coding for putative peptideshavebeen isolatedandcharacterized. Theyare the real killers.Antivenomproduced against themajor Na-ScTx is capable ofneutralizing the entire symptoms of intoxication of experi-mental animals. The K-ScTx are shorter peptides containingfrom 22 to 42 amino acid residues, mostly packed by 3 or 4disulfide bridges. Both the Na- and K-ScTxs have a tridimen-sional folding that comprises a short alpha-helix segmentand a triple anti-parallel beta-sheet structure. Four sub-classes of K-channel specific peptides were identified: a, b, gandk-peptides (Tytgatet al., TrendsPharmacolSci 20:445-47,1999). They usually block the channel by binding to themouth of the channel. They are not real killers, althoughproduce abnormal depolarization of cells, which causesuncoordination of excitable tissues, but also modulate somephysiological functions. Calcins are Ca2þ-channel specificpeptides that affect mainly Ryanodine sensitive calciumchannels, but also can affect voltage-gated Ca2þ-channels




(Valdivia et al., PNAS 89:12185-89, 1992; Olamendi-Portugalet al., BBRC299:562-68, 2002). Chorotoxin is theonly peptideknown to impair chloride permeability. Scorpion venomsmight also have cardiotoxins, hemolytic toxins, antimicrobialpeptides, enzymes (hyaluronidase, acetylcholinesterase,phospholipase, metalloproteinase and sfingomyelinase D)that cause intoxication. Many other components are presentin scorpion venom, from 100 to 600 different molecularmasses have been identified in their venoms by mass spec-trometry analysis, but for some of them, the structure andfunction are still unknown; others are being used for theirpossible therapeutics applications, such as: antibiotics andimmunomodulators or analgesic substances.

Acknowledgementssupported in part by NIH grant (R01-HL108175), DGAPA-

UNAM grant IN204110 and Instituto Bioclón S.A. de C.V.

Keywords: ion-channel; scorpion; toxin10.1016/j.toxicon.2012.04.176

176. Is the Endogenous Opioid System Involved in theAntalgic Effect of Scorpion Toxins in Mice?

Marie-France Martin-Eauclaire 1,2, Najwa Abbas 1,2,Pierre E. Bougis 1,2, Régis Guieu 3

1Aix-Marseille University, CNR2M, 13015, Marseille, France2CNRS, UMR 7286, Marseille, France3 Laboratoire de Biochimie Centre, Centre Anti Douleur, AP-HM, CHU Timone,Marseille, FranceE-mail address: [email protected] (M.-F. Martin-Eauclaire).

Background: We have hypothesized that pain reliefinduced by scorpion toxins may be the result of a counterirritation phenomenon due to the activation of “diffusenoxious inhibitory control”. We have examined the alpha-and beta- scorpion toxin effects on the mice behavior duringnociceptive tests, after peripheral administration. In partic-ular, we have analyzed the involvement of the endogenousopioid systems in the antinociceptive effects observed afterinjections of the alpha-anatoxin AmmVIII, aweakmodulatorof Nav1.2 channel (the muscular Nav1.4 channel remainsalmost insensitive to its application), and of the depressantinsect-selective beta-toxin LqqIT2 previously shown to bedevoid of neurotoxicity for mammals. They could be effica-ciously injected in mice, even in large amount, withoutinduction of any apparent toxic symptoms.

Methods: The analgesic effects of the two scorpiontoxins, administered in mouse at different doses (12 miceeach dose) by intraperitoneal route, were observed onhot plate and tail flick latencies. We then compared theseeffects with those obtained after injection of well-knowncommercial antinociceptive drugs (ketoprofen andDAMGO) or after acetic acid administration or cold-watertail immersion. These two last tests served as counterirritation tests and are well-known to induce pain reliefthrough the activation of the diffuse noxious inhibitorycontrol (DNIC) and the release of endogenous opioids. Wealso evaluated the effects of toxins and acetic acid onspinal cord c-Fos mRNA expression, a proto-oncogene,which increases after painful stimulus.

Results: Both toxins increased latencies in a dosedependent manner. Also, an increase in latencies obtainedwith toxins, acetic acid, or cold-water tail immersion waspartly reversed by the co-administration of naloxone, a mopioid receptor antagonist. AmmVIII, LqqIT2, or acetic acidinjected alone inducedan increase in c-FosmRNAexpression.

Discussion: The antalgic effects observed after admin-istration of scorpion toxins could be partly due to a counterirritation phenomenon that implicates the activation of anendogenous opioid system. The toxins could first producea small dorsal root ganglion neurons (DRG) hyperexcit-ability which, in a second time, could lead their Navchannels to steady-state inactivation and a decrease in theneuron signaling involved in pain pathways.

Conclusion: To find a pharmacological answer tothese observations, we have now to explore the Amm VIIIand Lqq IT2 effects, as well as those of the highly lethal“classical” alpha- or beta- toxins, on the voltage-gatedsodium currents expressed in DRG neurons.

Keywords: pain, scorpion toxin, analgesia10.1016/j.toxicon.2012.04.177

177. IgY Antibodies Anti-Tityus caripitensis Venom:Purification and Neutralization Efficacy

Alvarez O. Aurora 1,2, Montero Yuyibeth 1,Jimenez Eucarys 1, Zerpa Noraida 1, Parrilla A. Pedro 2,Malave Caridad 1

1 Laboratorio de Biciencias, Instituto de Estudios Avanzados, Caracas,Venezuela2 Laboratorio de Farmacologia, Universidad de Oriente, Bolivar, VenezuelaE-mail address: [email protected] (A.O. Aurora).

Background: Tityus caripitensis is responsible for mostaccidents due to scorpion stings in northeastern Ven-ezuelan regions. Avian antibodies (IgY) isolated fromchicken egg yolk represent a new alternative to be appliedas antivenom theraphies. In this work we produce IgYantibodies against Tityus caripitensis scorpion venom andevaluate its neutralizing capacity both "in vitro" and "invivo".

Methods: The anti-scorpion venom antibodies werepurified by precipitation techniqueswith polyethylene glycoland evaluated by MABA, an indirect ELISA, and western blotassays. The neutralization of lethality was evaluated by pre-incubation of venom together with antivenom prior totesting.

Results: The specificity of IgY antibodies was demon-strated by a dose-dependent inhibition in western blotassay when antibodies pre-absorbed with the venom didnot recognize the venom proteins from T. caripitensis. Theantivenom was effective in neutralizing 2LD50 doses ofT. caripitensis venom in vivo (97.8 mg of IgY neutralized 1mg of T. caripitensis venom).

Conclusion: Our results support the future use of aviananti-scorpion T. caripitensis venom as an alternative toconventional equine antivenom in our country.

Keywords: Tityus caripitensis, anti-venom, IgY antibodies10.1016/j.toxicon.2012.04.178




178. Development of Nanobodies and Derivatives withHigh Neutralizing and Protective Capacity againstScorpion Envenoming

Balkiss Bouhaouala-Zahar 1,2, Issam Hmila 1,Rahma Ben Abderrazek 1, Serge Muyldermans 3,Mohamed El Ayeb 1

1Venoms ans Toxins Laboratory, Institut Pasteur Tunis, Tunisia2Medical School of Tunis, University Tunis-El Manar, Tunisia3VIB Department of Structural Biology, Vrije Universiteit Brussel, BelgiumE-mail address: [email protected] (B. Bouhaouala-Zahar).

Background: Envenoming following scorpion stingis a public health issue in many parts of the world.During scorpion envenoming, highly toxic small poly-peptides of the venom diffuse rapidly within the victim,causing serious medical problems. Nanobodies (Nbs), therecombinant single-domain antigen-binding fragments ofcamel-specific heavy-chain only antibodies, offer specialadvantages in therapy over classic antibody fragments due totheir robustness and smaller size matching the size of thescorpion toxins (7 kDa). Our aim was to develop antivenomproduct which more quickly reach the highly diffusiblescorpion toxins.

Methods: We immunized dromedaries with toxins fromAndroctonus australis hector (Aah) scorpions and cloned thesingle-domain antibody fragments or nanobodies (15 kDa)from their B cells. Nanobodies against the two most toxiccompounds of the venom (AahI and AahII toxins) wereretrieved from the libraries, and their neutralization andprotective capacity was monitored in mice. Subsequentbispecific and humanized derivatives have been designedand their pharmacokinetics has been studied

Results: It is demonstrated that the retrieved Nano-bodies fully protected mice against high lethal doses(7 to 100 LD50) of scorpion toxins administered intra-cerebroventricularly. Moreover, where current antivenomfailed completely to neutralize 2LD50 of crude venominjected subcutaneously, the designed bispecific NbF12-10against AahI'/AahII toxins succeeded in neutralizing 5 LD50.Finally, the maximally humanized version of nanobodymaintains its high affinity for the toxin without concedingmuch on expression yield and stability.

Discussion: The bispecific NbF12-10 is the best candidateto develop a therapy in human against themost toxic venomcompound of one of the most dangerous scorpions. Ina challenge assay in which mice were subcutaneouslyinjected with a lethal dose of scorpion venom, the subse-quent intravenous injection of 85 microg of NbF12-10 pro-tected all mice, even if the whole procedure was repeated 3times. Furthermore, the NbF12-10 remained fully protectivewhen mice with severe signs of envenoming were treateda few minutes before the untreated mice died.

Conclusions: Nanobodies - derived from HCAbs ofcamelids and selected after phage display - show greatpotential to provide a more efficient therapy against scor-pion envenoming.

Keywords: nanobodies, scorpion toxins, protective capacity10.1016/j.toxicon.2012.04.179

179. Automated Mass Fingerprinting of ScorpionVenoms in the Nanogram Range

Marie-France Martin-Eauclaire 1,2, Pierre E. Bougis 1,2

1Aix-Marseille University, CNR2M, 13015, Marseille, France2CNRS, UMR 7286, Marseille, FranceE-mail address: [email protected] (P.E. Bougis).

Background: Several proteomic approaches wereemployed recently to assess the diversity of the contentof animal venoms. All involve techniques of liquid chro-matography and mass spectrometry. But, at some pointa tedious manual work of data processing must be per-formed. We have investigated the possibility to automati-cally produce a comprehensive venom mass fingerprint(VMF) from nanogram quantities of scorpion venom. Aworkflow was designed using the most recent advanceson ultra-high pressure liquid chromatography (UHPLC)coupled to an automated MALDI-TOF data acquisition anda specially designed data-processing.

Methods: Scorpion venoms were extracted by manualstimulation and constituted polled or individual samples.Nano UHPLC analysis were performed using a DionexUltiMate� 3000 RSLCnano system and 100 mm x 250 mmC18-reversed phase PepSwift Monolithic Nano PS-DVBcolumn. Direct on-line effluent spotting was performedusing a LC-Packing Probot and a sampling rate of 3 spotsper second with addition of 0.5 ml of twice-diluted CHCA-saturated matrix. MALDI-TOF spectra were recordedon a Bruker Ultraflex II automatically on each spot by usinga fuzzy logic feedback control system. A specially designedExcel-macro was used to filter for doubly charged or dimerions and to combine each individual peak list to a oneincluding the chromatographic retention time. A clusteringprocess was allowed to end with a single list of uniquemasses at a minimum threshold intensity.

Results: The optimal venom quantity to be injected onthe nano column was first determined. Few tens of nano-grams were the optimum and saturation was observed forhundreds. Special attention was given to the reproduc-ibility of the workflow. Statistics were performed onrepetitive sample injections and m/z relative error andnormalized relative intensity error were assessed. A verylow dispersion for mass value was found, which is crucialfor VMF comparison. Dispersion concerning peak intensi-ties was not so helpful, but not surprising sinceMALDI-TOF/MS is known to be not a good quantitative technique. First,we validated our automated VMF acquisition process usingthe well known Androctonus mauretanicus venom. Then,wewere able to study the venom of rare Buthidae collectedin Southeast France.

Conclusions: Taking into account the results of ourstudy, it is noteworthy that from tens of venom nanograms,comprehensive VMF can be achieved in a timeless and anautomatic way. This workflow could help us to considerphylogenic studies based on clades defined from suchcomprehensive lists of individual masses.

Keywords: UHPLC, venom mass fingerprint10.1016/j.toxicon.2012.04.180




180. Increased Incidence of Tityus trivittatusEnvenoming in the City of Buenos Aires

Guillermo Blanco 1, Rodrigo D. Laskowicz 2, E. EduardoScarlatto 1, Natalia Casas 3, Vanessa Costa de Oliveira 3,4,Laura C. Lanari 2, Néstor R. Lago 4, Adolfo R. de Roodt 2,41 Servicio de Toxicología del Hospital de Clínicas “José de San Martín” -Laito-CONICET, Argentina2Área Investigación y Desarrollo, Instituto Nacional de Producción deBiológicos, Administración Nacional de Laboratorios e institutos de Salud,Argentina3 Programa de Zoonosis, Ministerio de Salud de la Nación, Argentina4 Laboratorio de Toxinopatología, Centro de Patología Experimental yAplicada, Facultad de Medicina, ArgentinaE-mail address: [email protected] (A.R. de Roodt).

Background: Tityus trivittatus is the scorpion of highestmedical importance in Argentina. It is present in themajority of the big cities of the country favouring thecontact man – scorpion due its characteristic of adapting tolive in human constructions and environments. In recentyears, both the number of scorpion sighting reports, as wellthe number of envenomings increased in the country. Weundertook this study to determine if the population ofTityus trivittatus has expanded its geographical area duringthe last decade in the city of Buenos Aires and whether ornot there has also been an increase in the frequency ofenvenoming cases within the affected geographical areas.

Methods: the reported cases recorded in our institu-tions during 2001-2011 were classified by date and loca-tion, further geo-referenced in a digital map and analyzedin a geographic information system (GIS). Reported caseswere modeled as points and buffer areas were created 250m around each case to define an arbitrary measure ofgeographical influence. When reported Tityus eventsoccurred close to each other (less than 500m), overlappingareas became merged into a single area containing allpoints representing those events. These areas were differ-entially plotted for increasing time intervals from 2001 to2011. The total area associated with reported findings insquare km and the number of events per square km wascomputed for each time interval.

Time interval(Years)

CompromisedArea (km2)

Reportedfindings

Reportedfindings/km2

2001-2002 0.78 16 20.492001-2004 2.43 60 24.682001-2006 3.03 82 27.082001-2008 3.63 126 34.682001-2011 6.00 251 41.81

Results:Discussion: The geographical area within the city of

Buenos Aires, where some Tityus trivittatus findings havebeen reported, has increased at about 0.5 km2 per yearsince 2001. The incidence of sightings of scorpionscomputed by area has also consistently increased since2001, suggesting the presence of the species has becomepermanent. Despite the increasing number of sightings ofTityus trivittatus, the number of envenoming cases inBuenos Aires city did not increase in the last five years.

There have been no deaths and only one moderateenvenoming occurred in the city. The toxicity of thevenom of these scorpions in the city of Buenos Aires islower regarding that from other regions of the country.However, the increased geographic area where thesescorpions can be found, indicates the need to focusattention to prevent potential envenomings in zones ofthe city where the presence of T. trivittatus has not beenhistorically registered.

Keywords: Tityus trivittatus, scorpion, epidemiology, finding, Argentina10.1016/j.toxicon.2012.04.181

181. Evaluation of a Four-Hour Endpoint for Use inScorpion Envenomation Studies in Morocco

Rachida Soulaymani-Bencheikh 1,2,Emmanuelle F. Mangin 3, Asmae Khattabi 4,Zachary T. Fellows 5, Leslie V. Boyer 3

1 Poison Control and Pharmacovigilance Center of Morocco, Rabat, Morocco2University Ibn Tofail, Faculty of Science, Laboratory of Genetics andBiometrics, Kenitra, Morocco3VIPER Institute, University of Arizona, Tucson, AZ, USA4National Institute of Health Administration, Rabat, Morocco5Ross University, School of Medicine, North Brunswick, NJ, USAE-mail address: [email protected] (E.F. Mangin).

Background: Scorpion stings in Morocco are a signifi-cant public health issue and children under the age of 15are the most severely affected. The Moroccan poison center(Centre Anti Poison duMaroc, CAPM) uses a systematic fourlevel envenomation classification system. Scorpion anti-venom in North Africa has been controversial in the pastand is not currently in use in Morocco. The objective of thisstudy was to characterize a population for which effectiveantivenom treatment might have the greatest impact andto characterize potential endpoints for use in a subsequentprospective scorpion antivenom trial.

Methods: This was a retrospective review of CAPMrecords representing patients admitted for scorpion enve-nomation across Morocco. Patients included in the studywere 6 months to 10 years old, admitted for a scorpionsting between March 2007 and November 2009. Patientspresenting to the hospital more than 4 hours after a sting orwith an envenomation class IIa or below were excluded.Indicators of patient outcome were observed hourly for thefirst five hours after admission seeking evidence of changefor these parameters during that time. Final patientoutcome at hospital discharge was recorded. No patientreceived antivenom.

Results: Out of 349 cases, 244 met the studyinclusion and exclusion criteria. 18.4% (n ¼ 45) of thepatients progressed to a class III envenomation at sometime during their hospital stay. Out of 223 patients forwhom final outcome was available, mortality was 11.2%(n ¼ 25). Younger patients had the most severe clinicalsyndrome. Out of the 244 patients, only 3 (1.2%) hadclinical improvement documented within 4 hours ofadmission.

Discussion: Our findings are consistent with pastreports that scorpion envenomation syndrome without




antivenom persists for greater than 4 hours in North Africaand in North America. Recent clinical trials in NorthAmerica indicate that severe Centruroides envenomationresolves within 4 hours when promptly treated witheffective antivenom. Taken together, these findingssuggest that the four-hour endpoint in a similar pop-ulation could be used to test efficacy of an antivenomspecific to North African species.

Keywords: scorpions, pediatrics, endpoint determination10.1016/j.toxicon.2012.04.182

182. Two Case Studies of Pregnancy Outcomes afterScorpion Envenomation and F(ab')2 ScorpionAntivenom Treatment

Joanne M. Mallie 1, Sue Hoopmann 1,2, Janice A. Degan 1,Leslie V. Boyer 11VIPER INSTITUTE, University of Arizona, Tucson, AZ, USA2Chandler Regional Medical Center, Chandler, AZ, USAE-mail address: [email protected] (J.M. Mallie).

Background: The effects of venom and antivenomexposure during Case pregnancy have not been wellstudied. Clinical trials of scorpion antivenom recentlyincluded two pregnant women.

Case Studies: Between 2005 and 2011, 1970 patientswith presumed Centruroides envenomation wereenrolled and treated with an F(ab')2 scorpion antivenom ina multicenter treatment protocol in Arizona, USA. After-informed consent, subjects received 3-5 vials of Anascorp�,intravenous. Out of 1970 cases, 2 were eventually recog-nized as having been pregnant at the time of studyenrollment. Case 1 involved a 28-year-old woman pre-senting at 8 weeks’ gestation with nystagmus, tonguefasciculations and limb paresthesias. Aware that she waspregnant, medical staff reviewed the potential risks andbenefits of antivenom treatment with her before decidingto proceed in order to minimize the risk of envenomationitself. She received a total of 4 vials of antivenom, and hersigns and symptoms resolved 70 minutes after studyenrollment. At term she had a normal labor resulting indelivery of a healthy 9-lb boy. Case 2 was a 23-year-oldwoman who was unaware of pregnancy at the time of thescorpion sting. She presented with nystagmus, tonguefasciculations, chest heaviness, and limb paresthesias. Shereceived lorazepam, morphine and 3 vials of antivenom,with complete resolution of her symptoms in less than 2hours. One week later, she discovered that she was preg-nant; and she had a spontaneous abortion approximately22 days following the scorpion sting.

Discussion: Cause and effect cannot be proven by casedescription alone. It is clear that good pregnancy outcomeis possible with first trimester scorpion sting and F(ab’)2antivenom treatment; although pregnancy loss followingenvenomation and treatment for scorpion sting (here) orsnakebite (Langley, 2010) is also a consideration. Fetal lossas in Case 2 could result from unrelated causes; fromvenom interaction with maternal circulation, placenta orfetus; or perhaps from exposure to equine serum

derivatives. Neither of the patients in this series had signssuggestive of type 1 or 3 hypersensitivity; and the rapidresolution of systemic signs in both cases suggests thatantivenom use in systemic envenomation was likely morebeneficial than harmful.

Conclusions: Envenomated patients of childbearingpotential should be counseled as to risks and benefits priorto the administration of antivenom. The use of antivenomin many cases is likely to be a safer choice than supportivecare alone, but more evidencewill be necessary before finalconclusions can be made.

Keywords: envenomation, pregnancy, antivenom10.1016/j.toxicon.2012.04.183

183. Development of Immune Sera Against AlgerianScorpion Venoms: Which Antibodies for EnvenomingTreatment in Regions At-Risk?

Amina Ladjel-Mendil 1,2, Sonia Adi-Bessalem 1,2,Djelila Hammoudi-Triki 1,2, Fatima Laraba-Djebari 1,21University of Sciences and Technology “Houari Boumediene”, Laboratoryof Cellular and Molecular Biology, Faculty Biological Sciences, Algiers, Algeria2 Laboratoire de Recherche et de Développement sur les Venins, Route duPetit Staouéli, Algiers, AlgeriaE-mail address: [email protected] (F. Laraba-Djebari).

Background: The number of dangerous scorpion species(Androctonus australis: Hector: Aah, Androctonus ammourexi:Amx and Buthus occitanus tunetanus: Bot) raging in someregions At-Risk in the Maghreb remains a real concern forthe immune serum producers. The use of one or amixture ofantigens is still subject of controversy. In this order topropose an efficient immune serum three preparationswereundertaken, mono-, bi- and tri-valent were tested.

Methods: The efficiency of tri-valent serawas comparedto that of bi-valent and mono-valent one. Their effects oninduced tissue damage and inflammatory response wereevaluated

Results: Administration of tri-valent immune sera at 30min after envenomation, to animals injectedwith the Aah orAmx or Bot venom neutralized tissue damage (hemorrhage,edema, leukocyte infiltration) in myocardial tissue andhepatic parenchyma. Administration of these antibodytreatments also reduced themetabolical perturbations (CPK,LDH, AST, ALT, urea, creatinine and cholesterol) and bloodleukocytosis. However, hyperneutrophilia and eosinophiliainduced by the venomswere not significantly affected by theantibody treatments. These results showed also the similarefficiency of these three preparations against Aah venom,however, the tri-valent preparation presents a significantneutralizing effect than the mono-valent serum when Amxor Bot venom used for the envenomation. These results arevery competitive with those obtained by the already stan-dardized mono-valent immunotherapy.

Discussion: Administration of tri-valent antibodies maythus be beneficial in counteracting the whole pathophysi-ological effects induced by scorpion venoms. The efficiencyof this treatment could be due to the neutralization effectsof F(ab’)2 fragments on circulating of the three venoms.




Conclusions: It appears according to this study that thisimmune serum could be a great contribution to cover allthe polymorphisms existing in our country.

Table 1Effect of tri-valent antibodies (F(ab’)2 anti Aah, anti Amx and anti Bot) on metabolical perturbations induced by scorpion venoms.

CPK (IU/l) AST (IU/l) ALT (IU/l) ALP (IU/l) Urea (mmol/l) Crea (mmol/l)

Control 596.5 � 29.8 87 � 8.2 17 � 1.2 73.79 � 8 7.59 � 1 1.32 � 0.09Aah Venom 1546 � 77.3 192.6 � 15.3 56 � 5 184 � 15 10.8 � 0.9 16 � 0.8Bot Venom 1543 � 51.4 180 � 17 81.2 � 7 107 � 10 8.6 � 1 13 � 1.1Amx venom 2608 � 65.2 247.6 � 21.3 49.7 � 5 88 � 9 7.9 � 8 13 � 1.2Tri-valent-seraþ Aah venom 408 � 20.1 141.2�14 27.6 � 3.2 70 � 7 6.6 � 7.1 6 � 0.5Tri-valent-seraþ Bot venom 1098 � 36.6 163.7 � 15.3 22.7 � 1.8 68 � 7.1 8.2 � 0.95 4 � 0.3Tri-valent-seraþ Amx venom 934 � 31 132.8 � 10.2 44.6 � 5 61 � 5.9 6.8 � 0.5 12 � 1

TCPK: creatine phospho-kinase; AST: aspartate transaminase; ALT: alanine transaminase; ALP: alkaline phosphatise; Crea: creatinemia.

Fig. 1. Hepatic parenchymal and myocardial structure in envenomed miceand treated by trivalent-antibodies (Haematoxylin and eosin coloration,magnification 400, legends: H: hemorrhage, E: edema).

Keywords: antibodies, valence, scorpion venom, neutralization10.1016/j.toxicon.2012.04.184

184. Development of Novel Scorpion Anti-Venoms inMéxico

Baltazar Becerril, Lidia Riaño, Lourival D. PossaniDepartment of Molecular Medicine and Bioprocesses, Instituto deBiotecnologia, National Autonomous University of Mexico, Av. Universidad,2001, Cuernavaca, MexicoE-mail address: [email protected] (B. Becerril).

Background: Scorpions of the family Buthidae containvenoms that provoke severe envenoming to humans. Toxiccomponents are short-chain peptides that affect ion-chan-nels (being Naþ-channels the most important), causing anabnormal cellular function. Scorpions of the genus Centrur-oides usually have few highly toxic peptides in their venoms.We have shown that a monoclonal antibody (BCF2) wascapable of neutralizing the intoxication caused by both: toxinCn2 and the whole venom from the scorpion CentruroidesnoxiusHoffmann (Licea et al. Toxicon, 34, 843-847,1996). Thisfinding prompted the hypothesis that anti-venoms againstscorpions could be constituted by a few neutralizing anti-bodies. In order to confirm this idea, a library of single-chain

human antibodies (scFv) was constructed and evaluated bymeans of phage-display and directed evolution.

Methods: A selected scFv (3F) was evolved resulting ina neutralizing antibody (6009F) of the whole solublevenom of Centruroides noxius (Riaño et al FEBS J. 272:2591-2601, 2005; Patent US 7,381,802 B2).

Results: New scFv variants like 9004G havebeen generated. We demonstrated that 6009F and 9004Gare capable of protecting mice against venoms from twodifferent scorpion species: Centruroides suffusus suffususand Centruorides noxius. A key residue (F101VH) wasinserted into scFv 9004G lone improving its neutralizingcapacity. The scFv obtained (LR) is the best neutralizingantibody against main toxins and venoms of these twoscorpion species obtained to date by our group (Riaño et al.J. Biol. Chem. 286:6143-6151, 2011).

Conclusions: These results show that a single antibodymay have multiple neutralizing capacities. Following thesame strategy, several scFvantibodies capable of neutralizingthe main toxins of Centruroides limpidus limpidus have beenobtained. These results allow us to propose confidently thata cocktail containing a few optimized human scFv antibodieswill become the next generation of scorpion anti-venoms.

AcknowledgementsThis work has been partially supported by Instituto

Bioclón, S.A. de C.V., Mexico and DGAPA-UNAM IN204110 toLDP and CONACyT to BB.

Keywords: antivenom; phage display; scorpion; directed evolution10.1016/j.toxicon.2012.04.185

185. Safety of Equine F(ab’)2 Antivenom for ScorpionEnvenomation: Results of Prospective Clinical Trials

Leslie Boyer 1, Michelle Ruha 2, Jan Degan 1, Jody Mallie 1,Alejandro Alagón 3

1Venom Immunochemistry, Pharmacology, and Emergency Response (VIPER)Institute, University of Arizona, Tucson, AZ, USA2Banner Good Samaritan Medical Center, Phoenix, AZ, USA3 Instituto de Biotecnología, Universidad Nacional Autónoma de México,Cuernavaca, MéxicoE-mail address: [email protected] (L. Boyer).

Background: Use of serum products includingantivenoms is known to involve a risk of type 1 and type 3hypersensitivity. The technology involved in the produc-tion of serum derivatives has advanced during the pastcentury such that third-generation (“fabotherapeutic”)




products now have much higher potency relative to totalprotein content, and the quantity of immunoreactivematerial injected into patients has been in many casesgreatly reduced. Recent prospective clinical trials of equineF(ab’)2 scorpion antivenom provided an opportunity toconduct follow-up evaluations of safety outcomes with oneof these newer products.

Methods: Children and adults at selected sites in the USand Mexico, envenomated by scorpions between 2004 and2011, were eligible for enrollment in a series of fiveprospective clinical trials of AnascorpTM (Centruroides (Scor-pion) Immune F(ab’)2 (Equine) Injection). In all protocols,adverse events were prospectively monitored, includingspecific observation for symptoms suggestive of serumsickness during the two weeks following enrollment.Adverse events were categorized as to severity and possiblecause, all serious adverse events were individually reviewed,and descriptive statistics were applied to the results.

Results: 15 patients were enrolled in a double-blindprotocol, 7 of whom received antivenom. 78 patients wereenrolled in 3 open label protocols that mirrored the treat-ment group in the double-blind trial. 1425 additionalpatients were enrolled in a treatment protocol offered at 28hospitals across Arizona. The range of antivenom exposurewas 1-5 vials, intravenous. In the treatment protocol, 307adults and 1118 children were treated. Overall, 3 (0.2%) hadacute type 1 reactions and 8 (0.6%) appeared to have type 3reactions; but none had the full syndrome of classicalserum sickness. Therewere 30 serious adverse events, mostof which involved secondary respiratory consequences ofenvenomation or of excessive sedation concomitant withthe study. There were no deaths.

Discussion: Acute and delayed immune responses area risk with any serum derivative; but results of thesestudies show that refined third-generation fabother-apeutics can have remarkably low adverse event profiles, incontrast with historic descriptions of 60-90% rates of serumsickness using first generation antisera. This study was notdesigned to prove cause, but it is likely that this improvedsafety is a consequence of high potency (thus relatively lowprotein dose), lack of immunogenic Fc with the F(ab’)2fragment, and high purity of this preparation overall.

Keywords: safety, antivenom, hypersensitivity, serum sickness, clinical trial10.1016/j.toxicon.2012.04.186

N. Snakes

186. Effects of Captivity or Season on VenomComposition in Two Species of Rattlesnakes(Crotalus atrox and C. v. viridis)

Christopher J. Rex, Stephen P. MackessyUniversity of Northern Colorado, School of Biological Sciences, Greeley,CO USAE-mail address: [email protected] (C.J. Rex).

Background: Snake venoms consist of a variety ofbiochemical components that serve to immobilize, kill and

digest prey. Venom variability within snakes has typicallybeen attributed to factors such as age, season, environmentand diet, but studies exploring snake venom composition atfine scales of resolution (within individual snakes) areuncommon. Toexplore theeffects of several of thesevariables,two separate studies were performed: effects of captivity onthe venom composition of adult Western DiamondbackRattlesnakes (Crotalus atrox), and seasonal effects on venomcomposition in snakes collected during spring and fall (free-ranging adult Prairie Rattlesnakes, Crotalus viridis viridis).

Methods: Sixteen C. atrox were captured from CochiseCo., AZ and maintained in captivity for eight months ona diet of NSA mice. Thirty-three C. v. viridis were captured,PIT-tagged, and released in the spring and fall from twowell-defined den sites in Weld Co., CO. Venoms wereextracted shortly after capture and once every two-threemonths for the C. atrox. Venom samples from both studieswere subjected to reducing 1-D SDS-PAGE, reversed-phaseHPLC, MALDI-TOF mass spectrometry (MS), five differentenzyme assays and a fibrinogen degradation assay.

Results: For both studies, venom composition appearedto remain constant within individuals, as assessed by 1-DSDS-PAGE and fibrinogen digest assay results, while RP-HPLC and MALDI-TOF MS showed only minor differences.For C. atrox, venom L-amino acid oxidase (LAAO) andphosphodiesterase (PDE) activity significantly increasedover the course of captivity, with no changes occurring inmetalloproteinase (MPr), kallikrein-like serine protease(KLSP), or thrombin-like serine protease (TLSP) activities.Crotalus v. viridis venoms showed significantly higher PDEactivity and lower MPr activity in the spring than in the fall.Crotalus v. viridis venom TLSP activity levels also increasedsignificantly with time/age (from spring to fall and fall tospring), with no changes in LAAO or KLSP activity.

Discussion: Since the overall “fingerprint” for eachsnake's venom remained more/less constant, it can beconcluded that major changes in venom composition didnot occur within individuals in either species. Small butstatistically significant differences were, however, observedfor some venom enzyme activities.

Conclusions: These studies indicate that minor differ-ences in venom composition do occur in two rattlesnakespecies, as a function of season or captivity/diet. Althoughfurther testing will be necessary, these differences arelikely to be of minimal significance when treating cases ofhuman envenomation or producing antivenom.

Keywords: snake, venom, variation, Crotalus, atrox, viridis, season, captivity.10.1016/j.toxicon.2012.04.187

187. Metalloproteinases from Rear-Fanged (“Colubrid”)Snake Venoms: An Under-Utilized Resource forEvolutionary and Structure/Function Studies

Stephen P. MackessySchool of Biological Sciences, University of Northern Colorado, Greeley, CO, USAE-mail address: [email protected].

Background: Rear-fanged snakes (“Colubridae”) areabundant world-wide, and because their venom proteomesare typically much less complex than those of viperids and




elapids, they represent an excellent group for exploringquestions concerning the evolution and biological roles ofindividual venom proteins. Among the relatively low numberof protein families in snake venoms (w20), metal-loproteinasesare typical inmostcolubridvenoms,whilemanyother common protein families, such as LAOO, PLA2, serineproteinases and nucleases, are either uncommonly present orare wholly absent. Snake venommetalloproteinases (SVMPs)have major roles in pathogenesis following envenomationsand in the digestion of snake prey, and they are present in allclades of venomous reptiles; however, only a small number ofSVMPs from “colubrid” snake venoms have been purified andsignificantly characterized.

Methods: Venoms were extracted from many rear-fanged snakes using ketamine/pilocarpine as describedpreviously. Venoms were subjected to enzyme analyses,SDS-PAGE, MALDI-TOF-MS, and toxicity assays in severalanimal models. For select species, metalloproteinases werepurified fromcrude venoms using lowpressure LC andHPLC.

Results:Venoms from some species (Alsophis, Amphiesma,Coniophanes, Hydrodynastes) contain SVMP levels similar toviperid snakes, while others (Ahaetulla, some Boiga, Dry-mobius, Tantilla) contain levels comparable to elapids – low tobarely detectable activity. Alsophinase, a P-III SVMP fromAlsophis portoricensis venom, is a single chain protein witha mass of 56 kD. It contains potent fibrinogenolytic andhemorrhagic activities, and it induced subcutaneous andpulmonary hemorrhage in mice and Anolis lizards at doses of4 mg/g and higher. Sequence similarity analyses indicate thatmost colubrid SVMPs appear to be P-III proteinaseswith the 3domain structure typical of these metalloenzymes.

Discussion: SVMP activity is a common component ofmost colubrid venoms assayed, and while most appear to beP-III enzymes, a putative P-IImetalloproteinasewas describedfrom Rhabdophis tigrinus venom and a matrix metal-loproteinase-like enzyme was recently reported from thetranscriptome of Tachymenis. It is therefore clear that amongcolubrid snakes, one observes a diversity of classes of metal-loproteinases, but at present this diversity is poorly known.

Conclusions: Some general trends are identified frominformation currently available, and it is likely that the typeI/type II compositional dichotomy observed in rattlesnakevenoms may be reflected by a larger global pattern ofvenom composition in advanced snakes. Though “colubrid”metalloproteinases share some significant similarities withother SVMPs, different or novel hydrolytic specificitiessuggest they may be useful for structure/function studies.

Keywords: alsophinase, enzyme, toxin evolution10.1016/j.toxicon.2012.04.188

188. The In vitro Neurotoxic Effects of the NewlyDiscovered Central Ranges Taipan (Oxyuranustemporalis)

Carmel M. Barber 1, Peter Mirtschin 2, Nathan Dunstan 3,Terry Morley 4, Wayne C. Hodgson 1

1Monash Venom Group, Department of Pharmacology, Monash University,Victoria, Australia2 School of Pharmacy & Medical Sciences, University of South Australia, SouthAustralia, Australia

3Venom Supplies Pty Ltd, South Australia, Australia4Adelaide Zoo, South Australia, AustraliaE-mail address: [email protected] (C.M. Barber).

Background: The Inland Taipan (Oxyuranus micro-lepidotus) and Coastal Taipan (Oxyuranus scutellatus) arehighly venomous Australian elapids, whose venomscontain potent presynaptic (b) and postsynaptic (a)neurotoxins. Recently a new species of taipan, the CentralRanges Taipan (Oxyuranus temporalis), has been discovered(Doughty et al. 2007). Two specimens are held in captivityat the Adelaide zoo. The aim of this study was to charac-terise and compare the neurotoxicity of Central RangesTaipan venom with the other taipan venoms.

Methods: Central Ranges Taipan venom was obtained bymilking specimens housed at the Adelaide zoo prior to freeze-drying. Venoms from the Inland Taipan, Coastal Taipan,Papuan Taipan (Saibai Island) and Papuan Taipan (Merauke),for comparison, were supplied by Venom Supplies Pty Ltd.

Results & Discussion: Analysis of Central Ranges Taipanvenom using size-exclusion and reverse-phase HPLC indi-cated a markedly different ‘profile’ compared to the othertaipan venoms. In the chick biventer cervicis nerve-musclepreparation, Central Ranges Taipan venom (1 mg/ml) abol-ished indirect twitches (0.1Hz; 0.2ms, supramaximal V)witha t90 value of 24.3 � 4.0 min (n¼5). The venom also abol-ished responses to exogenous acetylcholine (1 mM, 30 s)and carbachol (20 mM, 60 s), indicating the presence ofpostsynaptic neurotoxins. Based on t90 values, the othertaipan venomswere found to be far less neurotoxic with thefollowing rank order of potency: Central Ranges Taipan (15.1� 2.0 min, n¼5, 3 mg/ml) > Inland Taipan (21.2 � 0.7 min,n¼3,10 mg/ml)> Coastal Taipan (81.4� 6.6min, n¼4,10 mg/ml) � Papuan Taipan Saibai Island (86.0 � 2.5 min, n¼3, 10mg/ml)> PapuanTaipanMerauke (129.3� 14.9min, n¼3,10mg/ml). The inhibitory effects of all taipan venoms(concentrations of either 3mg/ml or 10mg/ml) were neutral-ised by prior addition of CSL Taipan antivenom (3 Units/ml).

Conclusions: This study suggests that the venom of theCentral Ranges Taipan is highly neurotoxic in vitro andpotentially dangerous to humans.

References

Doughty P., Maryan B., Donnellan S.C., Hutchinson M.N.,2007. A new species of taipan (Elapidae: Oxyuranus) fromcentral Australia, Zootaxa, 1422, 45-58.

Keywords: taipans, neurotoxicity, antivenom10.1016/j.toxicon.2012.04.189

189. Neutralization Effect of Glycation on Myotoxic andNephrotoxic Effects Induced by BthTX-I

Veronica C.G. Soares 1,2,3, Camila L. Pires 1, Daniel Bristot 1,Henrique H. Gaeta 1, Daniela O. Toyama 4,Simone C.O. Buzzo 3, Selma D. Rodrigues 1,Marcos H. Toyama 1

1UNESP, Campus Experimental do Litoral Paulista, São Vicente, São Paulo,Brazil2UNIP, Institute of Health Sciences (ICS), Campus Jundiaí, São Paulo, Brazil3UNICAMP, IB, Campinas, São Paulo, Brazil



4University Presbiteriana Mackenzie, Center of Biological and HealthSciences (CCBS), São Paulo, São Paulo, BrazilE-mail address: [email protected] (V.C.G. Soares).

Background: Glycation, a chemical modification ofproteins with reducing sugars, indicating a possibleexplanation for the association between hyperglycemiaand the wide variety of tissue pathologies. A good exampleis glycosiled hemoglobin that can be used as a moleculartool to characterize the severity of diabetes. In this in vitrostudy, we investigated the effect of glucose, galactose andlactose on the structure and function of BthTX-I, which waschosen as the structural model of sPLA2. Glucose is knownand well characterized as one of the most important gly-cacanting agent of protein. Galactose is another mono-saccharide with the same functional groups found onglucose and lactose which is the dimer of galactose andglucose were also evaluated in this study.

Methods: The degree of glycosylation of BthTX-I withthe various glycating agents was performed using affinitychromatography phenylboronate, which allowed theidentification of glucose and galactose as twomain glycantsagents and purified the glycated BthTX-I form from the not.

Results: The analysis in reverse-phase HPLC showedthat both glucose and galactose significantly reducedhydrophobicity nature of BthTx-I. Moreover, both glucoseand galactose significantly changed the spectrum ofcircular dichroism and intrinsic tryptophan fluorescence ofBthTX-I. In addition, glucose and galactose also significantlyreduced edema, myotoxicity and nephrotoxicity induced bynative BthTX-I.

Discussion: These results indicate the susceptibilityof sPLA2 BthTx-I as non-enzymatic glycation by varioussugars. It also describes the effects of glycation on thestructure and pharmacological activity of BthTX-I.

Conclusion: Moreover, as BthTX-I is an example ofsPLA2 both in terms of structural and functional is possibleto conclude that nonenzymatic glycation might be affectother types of secretory PLA2 in diabetes for example.

Keywords: sPLA2, glycation, edema, myonecrosis.10.1016/j.toxicon.2012.04.190

190. First Draft of the Genomic Organization ofa PIII-SVMP Gene

Libia Sanz 1, Robert A. Harrison 2, Juan J. Calvete 1

1Consejo Superior de Investigaciones Científicas, Valencia, Spain2Alistair Reid Venom Research Unit, Liverpool School of Tropical Medicine,Liverpool, UKE-mail address: [email protected] (J.J. Calvete).

Review: The evolutionary pathway of snake venomtoxins remains poorly understood. The origin of SVMPs hasbeen inferred to have occurred following the split of thePareatidae from the remaining Caenophidians, approxi-mately 54-64 Mya, through recruitment, duplication, andneofunctionalization by positive Darwinian selection ofa closely related body cellular ADAM 7 or 28 ancestor gene.The evolutionary history of viperid SVMPs is repeatedlypunctuated by domain loss, and minimization of the

organization of genes coding for disintegrins, including lossof introns and coding regions, has been documented.However, details on the mechanisms of recruitment andmolecular events underlying the origin and evolution of theSVMP multigene family remain elusive. Moreover, thegenomic structure of any SVMP gene has not been reported,and thus the changes occurring after recruitment of theancestral gene into the venom gland remain hidden in thesnake genomes. Here we examine the gene structure ofthe 15652 bp Echis ocellatus pre-pro-EOC00089-like PIII-SVMP gene asssembled from PCR-amplified sequences ofoverlapping genomic fragments. The gene comprises 12exons interrupted by 11 introns. In a homologymodel of theEOC00089-like protein, the insertion of introns interruptingcoding regions lie between secondary structure elements.LINEs L2/CR1 and RTE/BovB, SINE/Sauria, and a hobo-acti-vator DNA transposon were identified within introns 1, 3, 7and 8. Pairwise amino acid sequence comparisons betweenEOC00089-like PIII-SVMP and its closest orthologs, humanand A. carolinensis ADAM28 showed that their ORFs share42%/59%, 49%/69%, and 48%/65% (identity/similarity),respectively. The protein-coding positions interrupted byeach of the 11 introns of the Echis PIII-SVMP gene areentirely conserved in theA. carolinensis andhumanADAM28genes. However, lizard and human ADAM28 genes contain 5introns not present in E. ocellatus. Strikingly, none of the 11introns shared between EOC00089-like PIII-SVMP and A.carolinensis ADAM28 genes exhibit conservation in size.Furthermore, Echis and Anolis introns exhibit quantitativelyand qualitatively distinctions in their inserted retroele-ments. Our finding that retroelements are exclusivelylocated within introns is consistent with the view thatintrons are added to genes presumably as transposableelements, and identifies introns as possible key elements inthe recruitment and amplification process of SVMPs into thevenom gland of extant snakes. Reptile genome sequencingprojects may shed light on this aspect of the emergence andevolution of venom toxins. Furthermore, the organization ofthe PIII-SVMP reported here provides a genomic explanationfor the emergence of dimeric disintegrin subunits encodedby short messengers.

Keywords: gene organization, SVMP, intronic retroelements10.1016/j.toxicon.2012.04.191

191. Dexamethasone Antagonizes the Myotoxic andInflammatory Effect of Bothrops Venoms

Fernando C. Patrão-Neto, Marcelo A. Tomaz,Marcos M. Machado, José Roberto Da S. Rocha-Junior,Paulo A. MeloLab. Farmacologia das Toxinas, ICB, CCS, UFRJ, Rio de Janeiro, RJ BrazilE-mail address: [email protected] (P.A. Melo).

Background: We investigated the toxic activities fromBothrops genus snake venom using in vivo and in vitroexperimental protocols in mice muscle and tested theprotector effect of dexamethasone (DEXA) in differentconditions, comparing it with the polyvalent antivenom.We also expanded the investigations on the antiophidiceffect of the Eclipta prostrata (EP) crude extract.





Methods: In vivo experiments were performed in mousemuscle and in mice with Bothrops jararaca and Bothropsjararacussu snake venom. We quantified the increase ofplasma creatine kinase (CK) activity as well the CK contentin the Extensor digitorum longus (EDL) of these animals. Wemeasured the edema and inflammatory response evaluatedby the presence of inflammatory cells at the inoculation sitewhenwe administrated B. jararacussu venom (1.0mg/Kg). Invitro we determined the increase of the rate of CK releasefrom the isolated EDL muscle perfused with appropriatednutrition solution. We also observed the amplitude of theindirect evoked twitch-tension at the isolated mousephrenic-diaphragm preparation.

Results: Treatment with DEXA (1.0 mg/Kg) preservedover 50.0 % of the muscle CK content in vivo when evalu-ated 24 and 72 hours after the injection of B. jararacussuvenom, and likewise decreased about 20.0 % of the edemainduced by this venom. DEXA reduced in 50.0 % the pres-ence of inflammatory cells in the muscle. The EP extract(50 mg/Kg) showed antagonized the edema and preservedthe muscle CK content, and its association with DEXAshowed an additive effect. EP also antagonized the increaseof plasma CK activity induced by the B. jararacussu venomin 77 %. The association of DEXAwith polyvalent antivenomdid not show additive or benefic effect. On the in vitroexperiments, DEXA did not show ability to antagonize theincrease of the rate of CK release from the muscles exposedto 25.0 mg/mL of B. jararacussu venom, neither to preventthe fall on amplitude of the indirect evoked twitch-tensionat the isolated phrenic diaphragm preparation, while the EPextract showed a 100.0 % protection at concentrations of 50and 100 mg/mL.

Discussion: Our results are showing that DEXA was ablein vivo to decrease the inflammatory response and did notshow any protective effect in vitro. Otherwise the inflam-matory responseswere almost completely neutralized by EP.

Conclusion: Our data together are demonstrating thatthe inflammation is an important element to be neutralizedon the envenomation by snake venoms.

Financial support: FAPERJ, CNPq.

Keywords: Bothrops venom, dexamethasone, myotoxicity, inflammation,Eclipta prostrate10.1016/j.toxicon.2012.04.192

192. Action of Venom Metalloproteinases on BasementMembranes: Pathogenesis of Hemorrhage andBlistering in Snakebite Envenomings

José María Gutiérrez 1, Teresa Escalante 1,Alexandra Rucavado 1, Jay W. Fox 2

1 Instituto Clodomiro Picado, Facultad de Microbiología, Universidad de CostaRica, San José, Costa Rica2Department of Microbiology, Immunology and Cancer Biology, University ofVirginia School of Medicine, Virginia, USAE-mail address: [email protected] (J.M. Gutiérrez).

Review: Zinc-dependent metalloproteinases are abun-dant components of snake venoms, especially in viperidspecies. Snake venom metalloproteinases (SVMPs) aregrouped in three classes (P-I, P-II and P-III), with various

subclasses, on the basis of their domain composition. SVMPsplay a key role in some of the most relevant pathophysio-logical manifestations of viperid envenomings, such ashemorrhage, blistering, necrosis and coagulopathy, andalso induce prominent inflammation. The pathogenesis ofhemorrhage by SVMPs is associated with the ability of theseenzymes to degrade proteins of the basement membrane(BM) and surrounding extracellularmatrix (ECM) in capillaryblood vessels. A combination of in vitro and in vivo experi-mental approaches has provided novel evidencefor understanding this complex phenomenon. Suchexperimental platforms include electron microscopy,intravital microscopy, immunohistochemistry, electropho-resis, Western blotting, and proteomic analysis of exudatecollected in the vicinity of affected tissue. Hemorrhagic andnon-hemorrhagic SVMPs of the P-I class have been comparedin order to identify differences thatmight help to understandthe basis of this pathological effect. In contrast to a non-hemorrhagic P-I SVMP, a hemorrhagic enzyme is able tohydrolyze perlecan and type IV collagen, as well as types VIand XV collagens, a finding that might have implications forits mechanism of action. On the other hand, P-III SVMPs are,in general terms, more potent hemorrhagic toxins than P-ISVMPs owing to the presence of exosites in their disintegrin-like and cysteine-rich domains, which enable these enzymesto bind to relevant targets in microvessels. Hydrolysis of keystructural components in capillary BMs and surroundingECM, with the consequent mechanical weakening of themicrovessel structure, provokes the distention of the capil-larywall, due to the action of hemodynamic forces operatingin vivo, a process that eventually brings mechanical disrup-tion of the vessel wall and extravasation. Similarly, the actionof SVMPs on BM components of the dermal-epidermalinterphase provokes the formation of blisters. Further studiesare necessary to compare the patterns of BM hydrolysisinduced by P-I and P-III SVMPs and to identify the structuraldeterminants of their highly variable toxicological profiles.

Keywords: snake, venom, metalloproteinase, hemorrhage, blistering10.1016/j.toxicon.2012.04.193

193. A Study of Venoms from Individual Snakes of TwoPopulations of Rhinocerophis (Bothrops) alternatus ofArgentina

Laura C. Lanari 1, Rodrigo D. Laskowicz 1,Vanessa Costa de Oliveira 2, Daniela Rocco 2,Néstor R. Lago 2, Roberto P. Stock 3, Adolfo R. de Roodt 1,21Área Investigación y Desarrollo/Serpentario, Instituto Nacional deProducción de Biológicos, Administración Nacional de Laboratorios eInstitutos de Salud, Ministerio de Salud de la Nación, Argentina2 Laboratorio de Toxinopatología, Centro de Patología Experimental yAplicada, Facultad de Medicina, Universidad de Buenos Aires, Argentina3 Instituto de Biotecnología de la Universidad Autónoma de México, MexicoE-mail address: [email protected] (A.R. de Roodt).

Background: Rhinocerophis (Bothrops) alternatus is thebiggest southernmost snake in the world and one of themost medical importances snakes in Southern SouthAmerica. In previous works we observed variationsbetween venoms of these snakes from different regions in




Argentina, and important individual variations in indi-vidual venoms from a region. In addition we could observethat despite some morphological differences in the snake-skin pattern, there were no morphological differences forsnakes of different isolated regions. For this reason, westudy the individual characteristics of the venom fromadult specimens of Rhinocerophis alternatus from two iso-lated regions, considering the amount of venom per animaland their toxicity.

Material and Methods: Specimens from Concordia,Entre Ríos (n¼ 17) and Olavarría, Buenos Aires (n¼ 17) werestudied. We determined the corporal size, venom yield,lethal (CF-1 Mice) and haemorrhagic (Wistar rats) poten-cies, and electrophoretic pattern by SDS-PAGE. Immuno-chemical reactivity of regional pools was studied usingexperimental specific antivenoms.

Results: The population under study was composed byanimals with a mean length of 94 cm (min. 76, max. 121),not showing differences between samples (p< 0.05).Venom extracted by animal (vacuum dry) was 152� 61mg,did not show differences between samples (p< 0.05).Electrophoretic pattern showed two main groups ofcomponents between 15-25 y 30-50 kDa, and major andminor components. Lethal potency of Concordia snakes(Md 64 ug; min. 44 ug, max. 149 ug) was greaterthan Olavarría snakes (Md 110 ug; min. 67 ug, max. 138 ug)(p < 0,05), with a mean potency of 13.9 LD50/mg y de 9.2LD50/mg of venom respectively. Haemorrhagic potency wasalso different in both samples. Concordia venom wasgenerally more hemorrhagic (MHD¼ 108 � 53 ug) thanOlavarría venom (202 � 94 ug) (p < 0.002). No strongrelationship between the hemorrhagic and lethal potencieswas observed. Immunological patterns were similar.

Discussion: Differences were observed between theactivities considered in both samples although with animportant dispersion of the values of toxic activities.Analysis of the individual venoms by SDS-PAGE under non-reducing conditions showed very similar patterns althoughsome bands were absent or weaker. The overall disparitiesin toxicity and venom composition could be due, amongother factors, to the variability in climate and food supplyunder which these populations have to live in two verydifferent regions. Variability of toxicity in individualsamples and their pools is discussed.

Keywords: Rhinocerophis alternatus, Bothrops alternatus, venom variability,toxicity, hemorrhage, lethality, electrophoresis10.1016/j.toxicon.2012.04.194

194. Effect of Heparin and L-Arginine on Skin TissueRegeneration after Cerastes cerastes Envenomation

Habiba Oussedik-Oumehdi 1,2, Fatima Laraba-Djebari 1,21University Sciences and Technology Houari Boumediene, LaboratoryCellular and Molecular Biology, Faculty Biological Sciences, Algiers, Algeria2 Laboratory of Research and Development on Venom, Pasteur Institute ofAlgeria, Algiers, AlgeriaE-mail address: [email protected] (H. Oussedik-Oumehdi).

Background: Tissue necrosis is a relevant local effect inViperidae envenomation, as it may lead to amputation.

The purpose if this study is to investigate the effect ofheparin and L-Arginine on tissue regeneration afterenvenomation.

Methods:Mice were intradermally injected in the dorsalskinwith a sublethal dose of Cerastes cerastes venom (30 mg/20 g of mice body mass). After 72 h, the dermonecroticactivity of the venom was assayed by macroscopic andhistopathological studies. For tissue regeneration assay, micewere humanely sacrificed at 1, 2, 3 and 4 weeks after i.d.injection of the venom. Heparin and L-Argininewere used astherapeutic drugs after envenomation. Mice treated withheparin, received an i.v. injection of heparin (10 mg/ g ofmicebody mass) at 15 min and 4 h after i.d. injection of thevenom. Mice treated with L-Arginine, were daily supple-mented with 3.75 mg/ ml of water, after i.d. injection of thevenom. Treated mice (with heparin and L-Arginine) weresacrificed at 1 and 2 weeks after envenomation.

Results: Results indicated that Cerastes cerastes venominduces a strong dermonecrotic activity with a minimumnecrotic dose of 19 mg/ 20 g of mice body mass. Histo-pathological study of skin sections, showed an alteration ofthe dermis, with hemorrhage, inflammatory infiltrationand a destruction of hair follicles and glandular structures.An acanthosis was observed in a disintegrated epidermisand oedema was also observed in the epidermo-dermicjunction. Macroscopic and histopathological study of skinregeneration indicated that skin tissue recovery occurredwithin 2 weeks after envenomation. Treatment of micewith heparin and L-Arginine, enhanced the capacity of skintissue regeneration, which was markedly observed only 1week post-envenomation.

Discussion: This study showed that Cerastes cerastesvenom induces an important alteration of skin structure,inducing dermonecrosis. Skin regeneration was improvedwhen animals were treated with heparin. Heparin is knownto be able to sequestrate growth factors and cytokines thatare implied in cell proliferation, fibroblast regeneration andangiogenesis. Skin tissue regeneration was also rescued byexogenous administration of L-Arginine, the precursor ofendogenous synthesis of nitric oxide. NO is an activator ofangiogenesis, that is involved in tissue wound healing.

Conclusion: These results suggest that heparin andpharmacological activators of the NO pathway mayconstitute, in association with immunotherapy, a rescuingtreatment to avoid loss in tissue mass and function inhuman envenomation.

Keywords: Cerastes cerastes, venom, dermonecrosis, regeneration,heparin, L- Arginine10.1016/j.toxicon.2012.04.195

195. A Novel Vascular Endothelial Growth Factor-LikeProtein from Gloydius tsushimaensis Venom

Hitomi Nakamura 1, Tatsuo Murakami 2,Takahisa Imamura 3, Michihisa Toriba 4, Takahito Chijiwa 2,Motonori Ohno 2, Naoko Oda-Ueda 1

1 Laboratory of Biological Chemistry, Department of Pharmaceutical Sciences,Faculty of Pharmaceutical Sciences, Sojo University, Kumamoto, Japan2Department of Applied Life Science, Faculty of Bioscience andBiotechnology, Sojo University, Kumamoto Japan



3Department of Molecular Pathology, School of Medicine, KumamotoUniversity, Japan4 The Japan Snake Institute, Ohta, Gunmma, JapanE-mail address: [email protected] (N. Oda-Ueda).

Background: Strong vascular permeability enhancingactivity ascertained by the Miles assay was found in Gloy-dius tsushimaensis venom when examined together withthe same amounts of the venoms of G. blomhoffii fromseveral regions of Japan and G.ussuriensis in South Korea.

Methods: In order to characterize this protein specificallyexpressed only in the venom of G. tsushimaensis of Gloydiusgenus in Far Eastern Asia, this protein was purified usingSuperdex75, MonoQ columns with ÄKTA purifier chroma-tography systems. The protein purified migrated on SDS-PAGE as a single band with molecular weights of 27 kD and13 kD under non-reducing and reducing conditions,respectively, suggesting that it exists as homodimer in nativecondition. The purified protein was digested with trypsinand the fragments were analyzed by MS on a ESI-Q-TOF.

Results: The sequences obtained fromMascot analysis ofthe MS data showed homology to other snake venom VEGFproteins. Since we found the conserved sequence in the 5’UTR for the snake venom VEGF cDNAs, we performed the 3’RACE using this conserved sequence as the 5’ primer and theadapter sequence attached to the 3’ oligo(dT) primer toacquire the full sequence ofG. tsushimaensisVEGF cDNA. ThecDNA was 487 bp long and had an ORF of 146 amino acids.

Discussion: The protein predicted showed about 80 %identity in sequence to previously published VEGF homo-logs from Viperidae snake venoms. Since the vascularpermeability enhancement by the protein purified wasinhibited by VEGF 2 receptor inhibitor but not by brady-kinin B2 receptor antagonist or histamine antagonist in theMiles assay, it is certain that the vascular permeabilityenhancement due to Gloydius tsushimaensis venom iscaused by its VEGF-like protein possibly through VEGF 2receptor. It has been reported that the toxicity (LD50 value)and hemorrhagic activity of G. tsushimaensis venom areabout one half and one hundredth those of G. blomhoffivenom, respectively.

Conclusions: The major activity of G. tsushimaensisvenom could be ascribed to its VEGF-like protein. This isthe first report of the VEGF-like protein from Gloydiusgenus.

Keywords: VEGF like protein, snake venom, regional evolution10.1016/j.toxicon.2012.04.196

196. Synthetic Peptides from Viperid Phospholipase A2Myotoxins: Small Structures with Diverse BiomimeticActions

Bruno LomonteInstituto Clodomiro Picado, University of Costa Rica, San José, Costa RicaE-mail address: [email protected].

Review: Phospholipases A2 (PLA2) present in thevenoms of viperid snakes are classified within the groupIIA of these small (w14 kDa) secreted enzymes. Althoughnot all viperid venom PLA2s display toxic activities, a large

group of isoforms with basic pI 's exert myotoxicityin rodent assays or cell culture models, and havebeen shown to be largely responsible for the necrosis ofskeletal muscle that frequently develops in human enve-nomings. Among these basic PLA2s, three types can berecognized: (a) the catalytically-active (Asp49) mono-meric or homodimeric PLA2 myotoxins; (b) PLA2s forminga heterodimeric complex (i.e., crotoxin) having bothneurotoxic and myotoxic activities; and (c) catalytically-inactive PLA2 homologues presenting the Lys49 substitu-tion, most commonly homodimeric, which display myo-toxic action. The highly conserved three-dimensional foldshared by both toxic and non-toxic group IIA PLA2srepresents a fascinating challenge for the study of struc-ture-function relationships, dictated by subtle variationsin surface-exposed amino acid residues that were selectedunder an accelerated evolution process. In the case ofmyotoxicity, at least two distinct mechanism of actionevolved in these proteins, that converge upon sarco-lemmal integrity impairment and subsequent cell death.Conventional Asp49 PLA2s appear to rely on their phos-pholipolytic activity to induce muscle damage, via theproduction of fatty acids and lysophopholipids. On theother hand, Lys49 PLA2 homologues, being catalyticallyinactive, have been shown to possess a specialized sitethat plays a key role inmyotoxicity. This functional site hasbeen mapped at their C-terminal region, and its exactidentity is still being refined by a variety of approaches.Interestingly, short synthetic peptide sequences corre-sponding to such C-terminal site of the Lys49 myotoxinsare capable of reproducing to some extent the diverseactivities of their parent proteins, thus acting as smallbiomimetics. These bioactive peptides present functionalproperties that might find useful medical applications, orserve as structural leads to develop improvements intheir pharmacological characteristics. In addition totheir (originally unforeseen) potential applications, thesepeptides also represent valuable tools for understandingstructure-function relationships in PLA2 myotoxins.

Financial support: Vicerrectoría de Investigación,University of Costa Rica (741-A9-513) and ICGEB (CRPProgram COS-08-03).

Keywords: phospholipase A2, myotoxin, biomimetics10.1016/j.toxicon.2012.04.197

197. Lupane Triterpenoids from Dipteryx alata Vogel asSnake Venom Inhibitors

Miriéle C. Ferraz 1, Marta M.D.C. Vila 1, José C. Cogo 2,Marcio G. dos Santos 3, Luiz M. Franco 4, Pilar Puebla 5,Arturo San Feliciano 5, Yoko Oshima-Franco 1

1University of Sorocaba, UNISO, Sorocaba, SP, Brazil2University of Vale do Paraiba, UNIVAP, S. J. dos Campos, SP, Brazil3 Federal University of Tocantins, UFT, Porto Nacional, TO, Brazil4Methodist University of Piracicaba, UNIMEP, Piracicaba, SP, Brazil5 Salamanca University, USAL, Salamanca, SpainE-mail address: [email protected] (Y. Oshima-Franco).

Background: The antiophidian properties of triterpe-noids have not been well-studied.





Methods: Triterpenoids from Dipteryx alata Vogel(lupeol), lupenone, 28-hydroxylup-20(29)-en-3-one,(28-OH-lupenone), and betulin, were pharmacologicallyassayed against Bothrops jararacussu (jararacuçu, Bjssu, 40mg/mL, n¼19) or Caudisona durissa terrificus (cascavel, CDT,10mg/mL, n¼10) snake venoms using a myographic apparatus.

Results: These venoms showed an irreversible neuro-muscular blockade in mice phrenic nerve-diaphragm prep-arations. However, the preincubated (30 min) mixture ofeach venom with each triterpenoid (1mg/5 mL) eliciteddifferent responseswhen added into the bath containing thepreparation. None of the triterpenoids caused neuromus-cular blockade at the chosen concentrations. All triterpe-noids were efficacious (p<0.05) in counteracting (in %) theblockade of Bjssu venom [betulin (68� 7)w lupeol (70� 8)> lupenone (45� 9)w 28-OH-lupenone (54� 5), n¼11, 5, 7and 4, respectively]; whereas only betulin (39.5 � 9, n¼9)and lupenone (49.5 � 8, n¼4) protected significantly theblockade-induced by CDT venom.

Discussion: The different mechanisms of action of bothvenoms can explain the superiority of these triterpenoidsagainst Bjssu venom, which is predominantly proteolyticand leads to a progressive myonecrotic process. Theseagents work less well against CDT venom, which ispredominantly neurotoxic.

Conclusions: Betulin is the best phytochemical inhib-itor against both snake venoms. The potential use of theseinhibitors as therapeutic agents in the treatment of snakebite envenomations, either alone or in combination withother therapies, needs to be evaluated in future studies.

Financial support: FAPESP 08/11005-5, CAPES/PROSUP;USAL, UNISO.

Keywords: Bothrops jararacussu, Caudisona durissus terrificus, Dipteryxalata10.1016/j.toxicon.2012.04.198

198. Isolation, Enzymatic Characterization and Action asSpreading Factor of a Hyaluronidase from Crotalusdurissus terrificus Snake Venom

Karla C.F. Bordon 1, Márcio G. Perino 1, José R. Giglio 2,Eliane C. Arantes 1

1Departamento de Física e Química, Faculdade de Ciências Farmacêuticas deRibeirão Preto, Universidade de São Paulo, Ribeirão Preto, SP, Brazil2Departamento de Bioquímica e Imunologia, Faculdade de Medicina deRibeirão Preto, Universidade de São Paulo, Ribeirão Preto, SP, BrazilE-mail address: [email protected] (E.C. Arantes).

Background: Snakebites related to the genus Crotalus areresponsible for the highest levels of mortality described forophidic accidents in Brazil. Hyaluronidase (Hyal) from Crota-lus durissus terrificus snake venom (CdtV) has shown to bea very active enzyme. However, studies on this enzyme,which is knownas “spreading factor”, are still limited. The aimof this studywas the isolation, enzymatic characterization, N-terminal sequencing and evaluation of Hyal from CdtV asa spreading factor of crotoxin and phospholipase A2 (PLA2).

Methods: The enzyme was purified through threechromatographic steps (CM-cellulose, Sephacryl S-100 andHeparin-Sepharose). Additionally, Hyal was submitted to

a reverse-phase C18 (0.46 cm x 25 cm) high performanceliquid chromatography (RP-HPLC) and its initial amino acidsequencing was performed by Edman degradation ina Shimadzu PPSQ-33A Protein Sequencer. The enzymaticactivity of fractions eluted from each chromatographic stepand the kinetic studies of Hyal were performed by turbi-dimetric assay. Sodium acetate buffers containing differentsalts (NaCl, KCl, CaCl2 and MgCl2) were employed todetermine ion effects on the enzyme activity. SDS-PAGEcontaining or not hyaluronan was run and periodic acid-Schiff stain was used to detect glycoprotein. Paw edemawas induced in male Swiss mice (28-32 g) by subplantarinjection of buffer containing crotoxin or PLA2 with orwithout Hyal, in the right hind paw. The volume of the pawwas measured with a paquimeter (Mitutoyo, Japan) beforeand after toxin injections at different intervals of time. Datawere analyzed by ANOVA and Student t-test.

Results: Hyal is a monomeric glycoprotein of 66.1 kDaand its first 25 N-terminal amino acids were sequenced. Itshowed maximum activity at 40 oC, pH 5.5 and in thepresence of 0.2 M NaCl. The soluble venom specific activitywas 145.1 turbidity reducing units (TRU)/mg, against5,065.9 TRU/mg for Hyal. Its Kcat was 3,780.95 min-1 forhyaluronan and about 400 min-1 for chondroitin sulphateA, B or C. The pure enzyme decreased the edema caused bysubplantar injections of buffer, crotoxin or PLA2.

Discussion: Hyal exhibited higher specificity to hyalur-onan than to chondroitin sulphate A, B or C. Divalentcations and 1M NaCl significantly reduced the enzymeactivity. Hyal increased the diffusion of crotoxin and PLA2

through mice tissues. Hyal potentiated crotoxin action,which was evidenced by mice death.

Conclusions: The reported purification procedure of theHyal from CdtV (0.02% protein recovery) was able toprovide a highly active antiedematogenic enzyme.

Financial support: CNPq, FAPESP

Keywords: Crotalus durissus terrificus, hyaluronidase, kinetic studies,isolation, antiedematogenic activity10.1016/j.toxicon.2012.04.199

199. BaPLA2-IV, an Acidic Phospholipase A2, IsolatedFrom Bothrops atrox Snake Venom: Biochemical andFunctional Characterization

Thales M. Junqueira, Lanuze G. De Toni, Adelia C.O. Cintra,Cássio P. da Silva, Suely V. SampaioFaculdade de Ciências Farmacêuticas de Ribeirão Preto - USP, Departamentode Análises Clínicas, Toxicológicas e Bromatológicas, Ribeirão Preto – SãoPaulo, BrazilE-mail address: [email protected] (C.P. da Silva).

Background: Snake venoms are rich sources ofcompounds displaying a large range of biological andpharmacological activities. As a rule, acidic Bothrops PLA2sdo not show experimental toxicity, but induce importantpharmacological effects, thus deserving further studies asan attempt to understand the correlation between struc-ture and catalytic/toxic functions.

Methods: Isolation was carried out by three chromato-graphic steps (molecular exclusion, ion exchange




and reversed-phase chromatography). The myotoxicactivity was analyzed using creatine kinase (CK) determi-nation and edema inducing assays in the subplantar regionof mice. BaPLA2-IV cytotoxic activity was evaluated byMTT,using HL-60, PC12, HEPG2 and B16F10 tumor lineages.

Results and Discussion: BaPLA2-IV represents about0.2% of Bothrops atrox whole venom. Its N-terminalsequencing showed similaritywith other PLA2-like enzymes(myotoxins) and PLA2s from Bothrops and Crotalus genera.BaPLA2-IV showed high phospholipase activity even at lowdoses, with 2 mg as a minimal dose, at different pHs andtemperatures, and anticoagulant activity at a dose of 1.5 mg,keeping plasma incoagulable for more than 60 min. Itshowed a low myotoxic activity when compared withBthTX-I, a basic Lys49 PLA2-like enzyme from B. jararacussusnake venom. For the edematogenic activity determination,the enzymes BaPLA2-IV, an acidic PLA2 from B. jararacussuand BthTX-I, as well as B. atrox venomwere used. When theedematogenic activity of BaPLA2-IVwas comparedwith thatof B. atrox venom (positive control – 100%), it correspondedto 80% (1h after injection) of the whole venom activity.BaPLA2-IV showed a low cytotoxicity in all treatments.

Conclusion: The acidic phospholipase BaPLA2-IV can beisolated by three chromatographic steps from B. atrox snakevenom. Comparison of its N-terminal sequence revealedsimilarity with other PLA2-like myotoxins (90%) and PLA2sfrom Bothrops (99%) and Crotalus (80%) genera, being anacidic Asp49 PLA2. It showed a high anticoagulant activityeven at low doses, low myotoxic and cytotoxic activitieson tumor cells, but a high edematogenic activity whencompared with BthTX-I.

Financial support: FAPESP, CNPq.

Keywords: acidic phospholipase A2, cytotoxicity, anticoagulant10.1016/j.toxicon.2012.04.200

200. Antinociceptive Potential of Raw Venom ofEgyptian Cobra and Black Tiger Snake: A FunctionalMagnetic Resonance Imaging Study

Susanne Wolz-Richter 1, Karl-Heinz Esser 2, Andreas Hess 1

1 Institute of Experimental and Clinical Pharmacology and Toxicology,Friedrich-Alexander University Erlangen-Nuremberg, Erlangen, Germany2 Institute of Zoology, University of Veterinary Medicine Hannover, GermanyE-mail address: [email protected] (S. Wolz-Richter).

Background: Pharmacological activities of animalvenom have been a focal point of interest in toxinology formany years. This study was performed to figure out if rawvenom of the Egyptian cobra Naja haje and of the Blacktiger snake Notechis ater niger produces antinociceptiveeffects in living animals.

Methods: First,we investigated the effects ofNaja haje andNotechis ater niger venom in rats using Hargreaves and tailflick tests for analgesic effects, rotarod test for motoricintegrity and open field test for locomotion. Our second andmain objective was to determine whether these anti-nociceptive effects can be measured by functional MagneticResonance Imaging (fMRI) using painful topical heat stimuli.In addition, we induced local hyperalgesia by injecting

Zymosan A into the left hind paw before the fMRI session.Consequently we could differentiate analgesic effects (rightpaw) and antihyperalgesic effects (left paw). fMRI measure-ments were performed on a 4.7 T Bruker BioSpec scannerusing a gradient echo EPI (Echo Planar Imaging) techniquewith 400 x 400 x 1000 mm spatial resolution throughout thewhole brain. Raw venom (Naja haje: 90 mg/kg; Notechis aterniger: 200 mg/kg) or NaCl (control) was injected intraperito-neally inside the scanner. Next a 100 min fMRI experimentwas performed. Blood oxygen level dependent (BOLD) signalsin the structures of the brainpainmatrixwere analyzed. BOLDamplitude and activated volume per brain structure werecalculated as fractional change with respect to baseline.

Results: A clear analgesic effect was found for Naja hajeand Notechis ater niger venom in behavioral tests: The pawand tail withdrawal latencies were significantly increased.Therewere no significant changes in rotarod and open fieldtest, indicating that there was no motor impairment due tothe neurotoxic activities of the venom. fMRI results showedthat Naja haje and Notechis ater niger venom significantlydecreased BOLD amplitude and activated volume in thal-amus, cortical structures, limbic system and basal gangliaduring nociceptive stimulation, showing differential anal-gesic and antihyperalgesic effects.

Discussion: Results of behavioral tests and decreases inneuronal activation throughNaja haje andNotechis ater nigervenom during nociceptive stimulation argue for analgesicand antihyperalgesic activity of these substances.

Conclusion: Using BOLD fMRI in anesthetized rats isa new valuable method to study antinociceptive effects ofvenoms without harmful sensations for the experimentalanimal. Raw Venom of Egyptian Cobra and Black Tiger Snakeshowed significant antinociceptive potential in this study.

Keywords: antinociception, BOLD-Imaging, fMRI rat brain, snake venom10.1016/j.toxicon.2012.04.201

201. Molecular Evolution of Snake VenomPhospholipase A2 (PLA2) Genes -Analysis of Group IPLA2 cDNAs from Venomus and Nonvenomous Snakes,and Lizards

Masahiko Yamashita, Tomonari Sawamoto, Toru TamiyaDepartment of Materials and Life Sciences, Faculty of Science and Technology,Sophia University, Tokyo, JapanE-mail address: [email protected] (T. Tamiya).

Introduction: Vertebrates universally have the group IBsecreted phospholipases A2 (sPLA2s) as digestive enzymes.The venomous snakes in Elapidae family have group IBsPLA2s and group IA sPLA2 as snake venom protein. Accord-ing to the sequence similarity of groups IA and IB sPLA2sgenes, these geneswere thought to beevolved fromthe sameancestral gene. In the erabu sea kraites, Laticauda semifasciata(Elapidae), two different types of mRNAs were transcribedfrom two different promoters on the same PLA2 genes. Onewas transcribed using a conventional promoter P1 on the 5’upstream region of the genes (4 exons and 3 introns) and theother was transcribed using the novel promoter P2 on intronI (3 exons and 2 introns). The amount of themRNAs encodingthe group IA sPLA2s transcribed from the P1 promoter (IA P1




mRNA)wasextremelyhigher than that fromtheP2promoter(IA P2 mRNA) in the venom glands of erabu sea karate. Wehave comparedP1andP2 IBmRNAs, andP1andP2 IAmRNAsfrom the venomous habu snake (Trimeresurus flavoviridis),the nonvenomous Japanese rat snake (Elaphe climacophora),and the Japanese grass lizard (Takydromus tachydromoides) ofReptilia Squamata, and analyzed how group IA PLA2sacquired the toxic function.

Result and Discussion: The deduced amino acidsequences of P2 IB cDNAs, the Japanese rat snakes, habusnakes and Japanese grass lizard had no signal peptides. Inaddition, the group IA P2 mRNAs from erabu sea krates andhabe snakes had the signal peptide encoding region. ExceptJapanese grass lizard, the nucleotide sequences of 5’ UTR ofthe P2 IB cDNAs from nonvenomous snakes, were verysimilar to the signal peptide encoding region of cDNAs fromerabu sea krait. Thus the IB P2 cDNAs without encodingsignal peptides originally had signal peptides encodingsequences. In the case of the Japanese grass lizard,the length of 5'UTR of the group IB PLA2 gene wasextremely shorter than that from the snakes examined. Asa consequence of mutations in a promoter region of thegroup IB P2 mRNA in the Japanese grass lizard, the tran-scription initiation sitemoved to downstream and 5’UTR ofP2 mRNA became very short. Thus the group IA PLA2 genemight possibly be found in the genome of erabu sea kraitsand habu snakes before the divergence of these snakes.

Keywords: evolution, snake, phospholipase A210.1016/j.toxicon.2012.04.202

202. Brazilian Coral Snake Phospholipases A2 InduceNeuronal Death in Primary Cultures of HippocampalNeurons

Nathalia Delazeri de Carvalho a,Raphael Caio Tamborelli Garcia d, Ivo Lebrun b,Durvanei Augusto Maria b, Silvia Carneiro c,Solange Castro Afeche a, Maria Regina Lopes Sandoval a

a Laboratory of Pharmacology, Butantan Institute, Av. Dr. Vital Brasil 1500,São Paulo, SP 05503 900, Brazilb Laboratory of Biochemistry and Biophysics, Butantan Institute, São Paulo,SP 05503 900, Brazilc Laboratoryof Cellular Biology, Butantan Institute, SãoPaulo, SP05503900, Brazild Laboratory of Toxicology Faculdade de Ciências Farmacêuticas daUniversidade de São Paulo, São Paulo, SP BrazilE-mail address: [email protected] (M.R. Lopes Sandoval).

Background: The venoms from Micrurus snakes(Elapidae) are endowed with pre- and postsynaptic neuro-toxins. Previously, we showed that the intrahippocampaladministration of the PLA2s neurotoxins, MlTx-8 and MlTx-9, isolated fromMicrurus lemniscatus venom induce seizuresand neuronal lesion. In the present work, we study theeffects of these neurotoxins on the cell death process incultured hippocampal neurons (CHpN).

Methods: Rat CHpN were dissociated from hippocampiof E18-E19 Wistar rat fetuses. Cultures were maintained at37�C in a humidified atmosphere of 5% CO2. On the seventhday, cells were incubated with MlTx-08 or MlTx-09 indifferent concentrations (0.74, 7.4 or 74nM) for 3, 6,12 or 24hours, depending on the experiment.

Results: The PLA2-neurotoxins had a potent time andconcentration-dependent neurotoxic effects on CHpN. Thecell viability determined through MTT assay indicated thatCHpN exposed for 12 hr to 74nMMlTx-8 or MlTx-9 reducedthe neuronal viability to 70,87�8,56% and 43,87�2,75%,respectively. This assay showed that these PLA2s-neuro-toxins reduce the mitochondria dehydrogenase activity. Toinvestigate whether mitochondria lay on themain pathwayunderlying the neurotoxicity, the mitochondria membranepotential (DJm) of CHpNs was evaluated by the fluores-cent probe Rodamine 123. After 3 hr of the CHpN stimu-lation with 74nM MlTx-8 or MlTx-9, we observeda reduction of the DJm in a magnitude of 26 and 39%,respectively. Inspection using fluorescent images of stain-ing with ethidium bromide and ultra structural analysis byscanning and transmission electromicroscopy showed thatmultiphase injury are characterized by overlapping celldeath phenotypes. Therewere both neurons with apoptosisand necrosis features observed: shrinkage, membraneblebbing, chromatin condensation, nucleosomal DNAfragmentation and the formation of apoptotic bodies. Themost striking alteration observed in the electron micros-copy was the fragmentation and rarefaction of the neuronprocesses network. Swollen terminal synapses, cell debrisand apoptotic bodies are observed among the fragmentedfibers. In the cytoplasm, it was noted numerous largevacuoles, swollen mitochondria and dilated Golgi.

Discussion: This is the first report showing that PLA2sisolated from the M. lemniscatus venom induce CHpN death.Ourmorphological data showedbothneuronswith apoptosisand necrosis features. We suggest that these neurotoxinscould act on mitochondria membrane permeability.

Conclusions: These neurotoxins will be a useful tool forunderstanding the death pathways in neurotoxic processesand contribute to elucidate the mechanism of action ofneurotoxic PLA2s.

Keywords: Micrurus lemniscatus , neuronal death, PLA2s-neurotoxins10.1016/j.toxicon.2012.04.203

203. Cellular and Humoral Immune Responses in HorsesImmunized with Crotalus Venom

Thaís R. Narcizo 1,2, Juliana J. Yamamoto 1,2,Mônica F. Silva 2,3, Ronaldo A. Ferreira 2,4,Sandra L. Moraes 2,5, Orlando G. Ribeiro 1,2,Olga M. Ibañez 1,2, José R. Marcelino 2,3,Mônica Spadafora-Ferreira 1,2

1 Laboratório de Imunogenética, São Paulo, Brazil2 Seção de Processamento de Plasmas Hiperimunes, São Paulo, Brazil3 Serviço de Imunologia, São Paulo, Brazil4 SeçãodeObtençãodePlasmasHiperimunes, InstitutoButantan, SãoPaulo, Brazil5 Instituto de Medicina Tropical de São Paulo, Universidade de São Paulo, SãoPaulo, BrazilE-mail address: [email protected] (M. Spadafora-Ferreira).

Background: The Brazilian Health Ministry reported that7.7% of 133,391 cases of snakebites between 2007 and 2011were caused by Crotalus rattlesnakes. The Crotalus durissusvenom is neurotoxic and myotoxic and can cause systemiccoagulation disorders. The specific antivenom antibody is the




only effective treatment in cases of accidents. Currently,Butantan Institute is responsible for 60% of the anti-crotalichorse serum produced in Brazil. Horses have been used foranti-venomproduction by their large size, resistance to toxinsand to the production of large amounts of plasma containingeffective IgG(T) antibody titles. Despite the well-establishedefficacy of the rattlesnake antivenom horse serum, there areno data in the literature about the specific cellular immuneresponses to the venom. The aimof this studywas to comparethe specific cellular and humoral immune responses to C.durissus venom in the peripheral blood of venom immunizedhorses.

Methods: Three groups of male horses (n¼15) wereimmunized according to a standard protocol for anti-cro-talic serum production consisting of different cycles ofimmunization with whole venom. Heparinized blood andserum samples were collected before and at differentperiods after immunization. Peripheral blood mononuclearcells (PBMC) were obtained by Ficoll-hypaque gradientseparation and stimulated in vitro with C. durissus venom.Proliferation was measured by thymidine [3H] incorpora-tion, after 5 days culture and considered positive whenproliferation index (IP) was > 2.0. Venom specific anti-bodies titers in sera were determined by ELISA.

Results: There was a significant cell proliferation in thedifferent PBMC samples, which increased after eachimmunization cycle. In addition, there was a variation inthe proliferation index among animals of the samegroup. All animals presented high titers of antivenom IgG(> 1: 2 x 105) with a fluctuation between immunizationcycles, and there was also a variability of the responseamong animals of the same group.

Discussion: Although, there was no individual correla-tion between the proliferative response and venom specificIgG(T) antibody titers for samples of the same period, therewas a correlation between theproliferative response and theIgG titers considering thewhole group of animals (p< 0.05).

Conclusions: The evaluation of different aspects ofcellular immune response like specific proliferative responseas well as cytokine production by immunized horses maycontribute to improve the protocol for immunization ofhorses for the production of serawith higher antibody titers.

Financial support: Fundação Butantan.

Keywords: Crotalus venon, horses, cellular immune response, antibodies10.1016/j.toxicon.2012.04.204

204. Vipera Lebetina Venom Nucleases andNucleotidases

Ene Siigur, Katrin Trummal, Külli Tõnismägi, Anu Aaspõllu,Jüri SiigurNational Institute of Chemical Physics and Biophysics, Tallinn, EstoniaE-mail address: [email protected] (J. Siigur).

Background: Snake venoms contain hundreds of bio-logically active components comprising enzymes (mainlyhydrolases), bioactive proteins and peptides. Amonghydrolases, nucleases and nucleotidases are ubiquitouslypresent in all snake venoms but there is almost no infor-mation about the amino acid and cDNA sequences of these

enzymes. Probably due to low abundance these enzymeshave not been found during proteomic studies of thevenoms. Snake venom nucleases are classified as endo-(DNases and RNases) and exonucleases (phosphodiester-ases - PDE). In addition, venoms contain 5`-AMP-hydro-lyzing 5`-nucleotidases (5`-NDases). The aim of this study isthe isolation of PDE, 5`-NDase and RNase from V. lebetinavenom and also amplification of these enzymes codingcDNAs from V. lebetina venom gland cDNA library.

Methods: The enzymes are isolated using gel filtrationon Sephadex G-100 sf, ion-exchange chromatography onCM-cellulose (PDE, 5’-NDase) and affinity chromatographyon DNA-agarose (RNase). Enzymes coding cDNAs areseparated, cloned and sequenced using standard methods.

Results: V. lebetina PDE is a nonspecific exonuclease withmolecular mass of 120 kDa and pI in neutral region (6.5-7.5).The pI heterogeneity is probably caused by different glyco-sylation of the enzyme (vast majority of snake venomenzymes are glycosylated). 5`-NDase has a molecular massabout 80 kDa and the pI in basic region (w9). Ribonuclease islocalized in the fourthgelfiltration fractionwithnervegrowthfactor and alkaline protease. Its molecular mass is w33 kDaandpIw10.PCRscreeningof theV. lebetinavenomglandcDNAlibrary resulted in isolation of 5`-truncated cDNAs encodingPDE and 5`-NDase. The PDE-encoding cDNA comprises 1704bp including 1644 bp ORF corresponding to 547-amino acidproteinsequence.ComparisonwiththeonlysnakevenomPDErepresented in the BLAST database, Crotalus adamanteus PDE,gives 95% identity in the cDNA and 91% identity in the proteinsequence. The 5`-NDase encoding cDNA comprises 1608 bpincluding 1215 bpORF corresponding to 404 amino acids. ThecDNA is 93% identical to ecto-5`-NDase of Gloydius blomhoffibrevicaudus, the protein sequence identity is 92%.

Conclusion: These results point to strong conservationof PDE and 5’-NDase in snake venoms.

AcknowledgementThe work is financially supported by ESF Grant Nos.

9458, 8899 and by the Estonian Ministry of Education andResearch Target Financing Grants Nos SF0690063s08 andSF0690029s09.

Keywords: snake venom, Vipera lebetina, phosphodiesterase, 5`-nucleotidase10.1016/j.toxicon.2012.04.205

205. A Novel Fluorometric Assay for the Evaluation ofSubstrates for Phospholipase A2 from the ColombianSnakes Bothrops asper and Crotalus durissus cumanensis

Andres M. Tibabuzo 1,2, Jackson Ocampo 2,Barbara H. Zimmermann 1, Chad Leidy 2

1Universidad de los Andes, Biochemistry and Molecular Biology of ParasitesGroup, Department of Biological Science, Bogotá, Colombia2Universidad de los Andes, Biophysics Research Group, Department ofPhysics, Bogotá, ColombiaE-mail address: [email protected] (A.M. Tibabuzo).

Background: Phospholipase A2 (PLA2) is a versatileenzyme present in all organisms. PLA2s in snake venom areresponsible for multiple systemic effects such as neuro-toxicity, myotoxicity, and hemolysis. They hydrolyze thesn-2 ester bond of phospholipids resulting in the formation




of lysophospholipids and free fatty acids. In this study weinvestigate the specificities of PLA2s in whole venomsamples of the Colombian snakes Bothrops asper and Cro-talus durissus cumanensis.

Methods: Crude venom samples were taken from B. asperand C. durissus cumanensis in the Guajira region of Colombia.The venom was lyophilized until use. Vesicles composed of1,2-di-O-octadecyl-sn-glycero-3-phosphocholine (DEPC)carrying thewater soluble fluorescent marker calcein at a selfquenching concentration (50mM) were used as reportervesicles, and target vesicles (substrate) made from 1,2-dite-tradecanoyl-sn-glycero-3-phospho-(1'-rac-glycerol) (DMPG),1,2-ditetradecanoyl-sn-glycero-3-phosphocholine (DMPC),1-hexadecanoyl-2-(9Z-octadecenoyl)-sn-glycero-3-phosphoe-thanolamine (POPE) or sphingomyelin (SM) were used formeasuring PLA2 activity through an indirect calcein releasemethod. We measured the release of calcein encapsulated inDEPC vesicles as triggered by lysophospholipids and free fattyacids produced by the action of PLA2s on target vesicles.Reporter vesicles were not hydrolyzed by the venom. Allmeasurements were performed in a pc1 Fluorometer (ISS,Urbana, IL) at 37�C with and without calcium (30 mM).Additionally, we evaluated vesicles simulating the outermembrane layer of platelets and red blood cells.

Results: Final concentrations of 0.5mg/mLwhole venomwere used in experiments with calcium and 10 mg/mL inabsence of calcium. At 37�C PLA2 activity was only observedwith DMPG vesicles, with C. durissus venom showing thehighest activity and lag time with calcium (%Release 47, lagtime 137 s). Experiments without calcium showed that C.durissus venom had again the highest activity (%Release 94)butB. asper venom showed thehighest lag time (71 s). DMPCvesicles showed activity near transition state temperature(24�C) with both venoms showing similar activity.

Discussion: Activity assays showed that PLA2 present inwhole venom has affinity for negatively chargedmembranes. No activity was observed with any of the othersubstrates including the vesicles that simulated plateletsand red blood cells, thus there must be other elements inthe membrane enhancing the affinity or the catalyticactivity of PLA2 in human cells

Conclusions: In this work we present a novel approachto study PLA2 activity in whole venom which will increaseunderstanding of its effect on the cell and may lead to thedevelopment of better treatments against snakebite.

Keywords: PLA2, liposome, fluorometric assay, enzyme activity10.1016/j.toxicon.2012.04.206

206. Isolation and Characterization of a D49Phospholipase A2 from Rhinocerophis (Bothrops)Ammodytoides venom

Herlinda Clement 1, Vanessa Costa de Oliveira 2,Fernando Z. Zamudio 2, Néstor R. Lago 2,Adriana Valdez-Cruz 3, Melisa Bernard Valle 2,Alejandro Alagón 2, Lourival D. Possani 2,Adolfo R. de Roodt 2,41Departamento de Medicina Molecular y Bioprocesos, Instituto deBiotecnologia, Universidad Nacional Autónoma de México, AvenidaUniversidad, 2001, Cuernavaca, Morelos, Mexico

2 Laboratorio de Toxinopatología, Centro de Patología Experimental yAplicada, FAcultad de Medicina, Universidad de Buenos Aires, Argentina3 Instituto de Investigaciones Biomédicas, México, Mexico4 Instituto Nacional de Producción de Biológicos, A.N.L.I.S. “Dr. CarlosG. Malbrán”, Ministerio de Salud, ArgentinaE-mail address: [email protected] (A.R. de Roodt).

Background: The snake Bothrops (Rhinocerophis)ammodytoides is the southernmost viper in the world. Itis generally a small snake which venom resemblesgenerally the characteristics of the venom from vipersof Bothrops genus. Although some studies were done onthe crude venom and its chromatographic fractions,there are not studies on the components of theirvenom. In this work we describe the isolation andcharacterization of a phospholipase A2 (PLA2) from thisvenom.

Methods: The enzyme was purified from the crudevenom by several chromatographic steps: Sephadex G-50,Mono-S in a FPLC system and C18 in a HPLC system and thepurity of this protein was verified by mass spectrometry.The toxic characteristics of the enzyme were studied bybiological assays.

Results: The enzyme represents the 3% of the totalmass of the venom. Close analysis of the sequence showsthat belongs to the D49 group of Type II PLA2. Theenzyme has 122 amino acids residues and the fullsequence was determined showing a very close simili-tude with a Botrhops erythromelas PLA2. The theoreticalmolecular mass was 13,867.65 Da, and the experimen-tally determined molecular weight was 13,853.65. Thedifference of 14 Da between these two values corre-sponds to the fact that the native enzyme is closelypacked by 7 disulfide bridges, accounting for this differ-ence. The isoelectric point is 6.13. The enzyme has lowtoxicity in mice. The LD50 was around 117 ug and theMMD was 200 ug. Killed mice showed pulmonarycongestion and some hemorrhagic spots in lungs and,hemorrhages in abdominal cavity represented by intra-peritoneal bleeding. Mice injected with 300 ug showedcongestion and acute edema of lungs with emphysema,atelectasis and thrombosis. The enzyme did not affect invitro or in vivo the coagulation although it was observedan inhibition of the clot retraction. A slight dose depen-dent inflammatory-edematogenic activity from doses of 2ug was observed. Although CK levels increased afterintramuscular injection, values were very close with thecontrols, despite its statistical significance (p < 0.05).Muscles showed lesions of different intensity dependingon the dose of enzyme injected, using 10 ug of enzymeafter 24 h muscles showed intense inflammation ofmononuclear type affecting vessels, inflammatory inter-fibrillar compromise and focal necrosis of muscularfibers.

Conclusions: The enzyme shows low toxicity althoughfurther studies are necessary to elucidate its relation withthe systemic bleeding observed at highest doses of theenzyme.

Keywords: Rhinocerophis ammodytoides, Bothrops ammodytoides, phos-pholipase, structure, toxicity10.1016/j.toxicon.2012.04.207



207. Developing of an Antielapidic Sera by Geneticimmunization

Henrique Roman Ramos 1, Inácio de Loiola MeirelesJunqueira de Azevedo 2, Carlos Chávez Olórtegui 3,Paulo Lee Ho 1

1Centro de Biotecnologia, Instituto Butantan, São Paulo, SP, Brazil2Centro de Toxinologia Aplicada, Instituto Butantan, São Paulo, SP, Brazil3 Instituto de Ciências Biomédicas, Universidade Federal de Minas Gerais,Belo Horizonte, MG, BrazilE-mail address: [email protected] (P. Lee Ho).

Background: Micrurus corallinus (coral snake) is a trop-ical forest snake belonging to the family Elapidae. Its venomshows a high neurotoxicity associated with pre- and post-synaptic toxins, causing diaphragm paralysis, which mayresult in death. In spite of a relatively small incidence ofaccidents, serum therapy is crucial for those bitten. However,the adequate production of antiserum is hampered by thedifficulty in obtaining sufficient amounts of venom froma small snake that is also difficult to keep in captivity.

Methods: Having recently finished the transcriptomicanalysis of M. corallinus, we have chosen five of the majorcomponents of the venom gland and synthesized over-lapping pentadecapeptides frameshifted by 3 residues,spanning the entire sequence of the five proteins by theSPOT method on cellulose membranes. The membraneswere then screened using an anti M. corallinus serum andpositive peptides were then used to design two multi-epitope genes that are being used for genetic andrecombinant immunization of female BALB/c mice. Fivedoses of 1 mg DNA will be administered to animals bybiolistic gene delivery. At the end of the immunizationperiod, animals will be bled and sera will be subjected tofurther analysis concerning its neutralizations capabilities.

Results: By the time of this abstract submission, the twomultiepitope genes were correctly synthesized and arebeing administered to animals. Further analysis concerningthe ability of these sera to neutralize the toxic effects of M.corallinus venom will be performed on the next days.

Financial Support: FAPESP, CNPq and FundaçãoButantan

Keywords: antielapidic serum, Micrurus corallinus, genetic immunization.10.1016/j.toxicon.2012.04.208

208. Fucose Moieties Are Essential for the Ability ofFucosylated Chondroitin Sulfate to Inhibit MuscleDamage Induced by Bothrops jararacussu Venom

Marcos Monteiro-Machado 1, Marcelo A. Tomaz 1,Marcelo A. Strauch 1, Bruno L. Cons 1, Hilmar D. Ricardo 1,Vinícius V. Martins 1, Roberto J. Fonseca 2, A.S. Paulo 1,2,P.A.S. Mourão 2, Paulo A. Melo 1

1 Laboratório das Toxinas e Substâncias Antagonistas, ICB, UFRJ, Rio deJaneiro, RJ, Brazil2 Laboratório de Tecido Conjuntivo, HUCFF, UFRJ, Rio de Janeiro, RJ, BrazilE-mail address: [email protected] (M.A. Tomaz).

Background: Snakebites by Bothrops jararacussu snakeinduces intense local tissue damage. Phospholipases A2 areenzymes present in the venom which are responsible for

a wide range of activities (Toxicon 45, p.1147, 2005). Somepolyanions have been shown to present antivenom prop-erties against this venom (Toxicon 31, p.285, 1993). A newnatural polyanion polysaccharide, named FucosylatedChondroitin Sulfate (fucCS), is involved in many biologicalactivities (JBC 282 (20), p.14984, 2007). We assessed theability of fucCS and its carboxi-reduced and defucosylatedanalogues to antagonize the muscle damage induced byB. jararacussu crude venom.

Methods: In vitro CK assays were performed with iso-lated mouse extensor digitorium longus (EDL) musclebathed with venom alone (25 mg/mL) or incubated withfucCS or analogues (10-50 mg/mL). In vivo experimentswere performed by i.m. venom injection alone or pre-incubated with fucCS or defucCS (1-10 mg/kg) and theplasma CK activity was evaluated before and 2 hours afterinjection (1 mg/kg). The phospholipase and hyaluronidaseactivities were measured using turbidimetric methods. TheCK content was evaluated in EDL muscle after a peri-muscular injection of venom (1 mg/kg). Histologicalsections were performed in EDL muscle after crude venomperimuscular injection. All experiments were approved bythe Committee of Animal Use of the Rio de Janeiro FederalUniversity (DFBCICB 026).

Results: It was observed that fucCS inhibits 75% ofphospholipase venom activity with IC50 ¼ 10 mg/mL (n¼10)and 100% of hyaluronidase activity with IC50 ¼ 7 mg/mL(n¼5), in concentration-dependent manner. Incubation offucCS with the venom eliminates the increase of plasma CK,in vivo (n¼4). The EDL muscle was preserved when exposedto venom with fucCS in vitro (30 and 50 mg/mL) (n¼4). Thereduction of the CK content was prevented by fucCS (1-10mg/kg) (n¼4). DeFucCS was unable to protect the phos-pholipase activity and miotoxicity in vivo and in vitro. Lightmicroscopy shows that fucCS can inhibit the muscle damageinduced by the venom (n¼4).

Conclusions: FucCS was capable to inhibit venomactivities related to tissue damage, although defucCS doesnot have this ability. These results indicate that fucCSpresents activity against Bothrops jararacussu venom andwe believe that this antivenom activity may be due to theinteraction of negative charges of fucose moieties of fucCSwith positively charges toxins present in this snake venom,like others polyanions.

Financial Support: CNPq, CAPES, FAPERJ and PRONEX.

Keywords: Bothrops jararacussu, fucosylated chondroitin sulfate,myotoxicity10.1016/j.toxicon.2012.04.209

209. Venom-Antivenin Binding and Neutralization ofVenom Toxicity: Application of Size-ExclusionChromatography to Assess Venom-Antivenin Binding

Charles G. SannyOklahoma State University - Center for Health Sciences, Dept. of Biochemistryand Microbiology, Tulsa, OK, USAE-mail address: [email protected].

Background: The World Health Organization isencouraging the development of alternative methods to





animal testing for evaluating venom toxicity and antiveninprotection against lethality. Size-exclusion chromatog-raphy (SEC) can provide an estimate of venom-antiveninbinding based on the formation of venom-antiveninimmune complexes, and could provide a priori informationuseful in antivenin protection studies. SEC analysis is rele-vant to antivenin protection if one assumes that venom-antivenin binding is required for protection. Venom-anti-venin binding, however, does not guarantee protection;therefore, both immune complex formation and neutrali-zation of venom toxicity (activity) need to be evaluatedwith comparable reactants.

Purpose: The purpose of this presentation is to furtherintroduce the concept of SEC analysis as an additional tool inassessing the relationships of venom-antivenin complexformation and neutralization of venom toxicity or lethality.Two articles that address these concepts have been pub-lished in Toxicon and are the primary sources of data withinthis presentation1, 2.

Method: 1) Venom, antivenin, and venom-antiveninmixtures are prepared. 2) SEC elution profiles are obtainedfor each sample. 3) Regions within the elution profiles arechosen for integration based on comparison of control andvenom-antivenin mixture profiles. 4) Profile region areasare determined for each sample. 5) Region area associatedwith venom-antivenin binding is estimated from thedifference between control and venom-antivenin mixtureregion area (i.e. D Area). 5) Concentration dependantchanges in D Area are fit to dose-response functions fromwhich D Areamax (maximum D Area) and EC50 (effectiveconcentration of reactants at one-half D Areamax) can beestimated.

Examples: SEC analysis. Interaction of Crotalis atrox(Western diamondback rattlesnake; C. atrox) venom andCrotaldae Polyvalent Immune Fab (ovine) antivenin(FabAV) illustrates the procedure for assessing venom-antivenin interaction. Concentration dependent changes inD Area for FabAV and C. atrox, C. varidis varidis (prairierattlesnake), Agkistrodon contortrix contortrix (southerncopperhead), and A. piscivorus leukostoma (westerncottonmouth) venom interactions are compared. Neutrali-zation PLA2 Activity. Concentration dependent changes in DArea and venom phospholipase A2 activity illustrateconcurrent assessment of venom-antivenin binding andneutralization of venom toxicity (activity).

Application: SEC analysis can be used to estimate andcompare EC50 (which inversely reflects venom–antiveninbinding affinity) and maximum D Area (which reflectsmaximum reactivity) of various venoms and antivenins(or other anti-venoms). SEC analysis parallel to venomactivity assays could estimate the concentration of anti-venins required for neutralization of venom toxicity(activity).

References

Price, J.A. and Sanny, C.G. Toxicon 49:848-854, 2007; 2.Sanny, C.G. Toxicon 57:871-881, 2011.

Keywords: venom, antivenin, SE-chromatography10.1016/j.toxicon.2012.04.210

210. Understanding the Preclinical Efficacy Profile ofAntivenoms: From the Lethality Potency Assay toAntivenomics

José María Gutiérrez 1, Bruno Lomonte 1, Guillermo León 1,Davinia Plá 2, Álvaro Segura 1, María Herrera 1,Mauren Villalta 1, Mariángela Vargas 1, Gabriela Solano 1,Libia Sanz 2, Julián Fernández 1, Yamileth Angulo 1,Juan J. Calvete 2

1 Instituto Clodomiro Picado, Facultad de Microbiología, Universidad de CostaRica, San José, Costa Rica2 Instituto de Biomedicina, CSIC, Valencia, SpainE-mail address: [email protected] (J.M. Gutiérrez).

Review: The great variability in the biochemical andtoxicological profiles of snake venoms poses a great chal-lenge to the selection of venom mixtures for immunizationof animals for the production of antivenoms, aswell as to thepreclinical testing of the neutralizing efficacy of antivenoms.Traditionally, the evaluation of antivenom potency has beenbased on the neutralization of lethal activity of venoms inmice. Although this test continues to be the gold standardfor antivenom potency assessment, advances made in thecharacterization of snake venoms have paved the way fora more in depth analysis of antivenom preclinical efficacy.Since the venoms of many snake species, particularly of thefamily Viperidae, induce a spectrum of toxic effects, evalua-tion of antivenoms should include the neutralization notonly of lethality, but also of additional toxic activities, such ashemorrhagic, edematigenous, myotoxic, coagulant, anddefibrinogenating effects. Moreover, analysis of theneutralization of some enzymatic activities, such asproteinase and phospholipase A2, has been also imple-mented. More recently, the proteomic characterization ofsnake venoms, i.e. ‘venomics’, has provided a highly detailedview of the complexity and variability of venom composi-tion. An adaptation of the proteomic platform to the analysisof immunoreactivity of antivenoms, a field known as ‘anti-venomics’, now brings the possibility of identifying venomcomponents for which antivenoms contain antibodies. Thecombination of in vivo and in vitro neutralization tests,together with antivenomics, allows for a detailed charac-terization of the reactivity of antivenoms against homolo-gous and heterologous snake venoms. These principles willbe illustrated for the case of the polyvalent antivenommanufactured in Costa Rica and used in Central America inthe treatment of envenomings by viperid snakes. This typeof integrated analysis can be of benefit tomanufacturers andregulatory agencies in various parts of the world.

Keywords: antivenoms, neutralization, potency test, antivenomics10.1016/j.toxicon.2012.04.211

211. Effect of Low Level Laser Therapy (LLLT) onBothrops jararacussu Venom-Induced Myotoxicity inMuscle Cells

Camila A.A. da Silva 1, Raquel A. Mesquita-Ferrari 1,José C. Cogo 2, Stella R. Zamuner 11Master and PhD program of Rehabilitation Sciences at the UniversidadeNove de Julho-UNINOVE, São Paulo, SP, Brazil



2 Laboratory of Physiology, Institute of Research and Development, Vale doParaíba University-UNIVAP, São José dos Campos, SP, BrazilE-mail address: [email protected] (S.R. Zamuner).

Background: Local myonecrosis is a common conse-quence in envenoming caused by snakes of the genusBothrops which occurs through the action of myotoxins thatacts directly in the muscle cell membrane. Antivenomtherapy and other first-aid treatments do not reverse thelocal myonecrose. Thus, there is an urgent need to findtherapies that can complement antivenoms in the neutral-ization of local tissue damage. The low level laser therapy(LLLT) is being considered as an alternative treatment formuscle injury situations because its bioestimulation effect.

Objective: The present work was designed to investi-gate the effect of LLLT in muscle cells submitted to injury byBothrops jararacussu venom (BjssuV).

Methods: C2C12muscle cell line was used. The cells weregrown in culture medium DMEM supplemented with 10%fetalbovineserum, incubatedat37�Cwith5%CO2for24hoursfor cell attachment, after that, the cells received BJssuVvenomin therespectiveconcentrations1,6,12.5,25and50mg/mLandincubated for 15, 30 and 60 min. The cell viability anddetachment were analyzed by MTT and crystal violet assay,respectively. Cells were irradiated for 13 s immediately afterthe venom administration with a semiconductor laser at 635and 830 nm, doseof 2.5 J/cm2 andpower of 100mW. The cellsthat did not receive venom and irradiation served as control.

Results: Our results showed that BjssuV causeda decrease in cell viability and detachment of C2C12 cellsthat was dose and time dependent. The venom causeda significant decrease in cell viability at doses of 12.5, 25 and50 mg/mL. Also, results showed an increase in thepercentage of cell detachment, this effect was morepronounced at doses 25 and 50 mg/mL promoting 100% ofdetachment cells at 30 and 60 min. LLLT increased cellviability by 86% and 92% in C2C12 myocytes by 635 and 830nm, respectively, 30 min after the venom administration.The laser had no effect on cell detachment caused by venom.

Discussion: BjssuV is toxic to muscle cells and LLLT wasable to protect the muscle cells against injury caused byvenom.

Conclusion: The use of LLLT should be considered asa potentially useful therapeutical approach for treatment ofthe local effects of Bothrops snakebites.

Keywords: muscle cells, myonecrosis, laser therapy, viability, cell detachment10.1016/j.toxicon.2012.04.212

212. Effect of Light Emitting Diode (LED) in theInflammatory Response and Myonecrotic Effect Inducedby Bothrops asper Snake Venom

Katia M.B. Moura 1, Ana M. Barbosa 1, Carlos J. Lima 2,José M. Guttiérrez 3, Stella R. Zamuner 41 Laboratory of Physiology, Institute of Research and Development, Vale doParaíba University, UNIVAP, São José dos Campos, SP, Brazil2 Laboratory of Optobiomedic, UNICASTELO, São José dos Campos, SP, Brazil3Clodomiro Picado Institute, Faculty of Microbiology, University of CostaRica, San José, Costa Rica4Master and PhD program of Rehabilitation Sciences at the UniversidadeNove de Julho - UNINOVE, São Paulo, SP, BrazilE-mail address: [email protected] (S.R. Zamuner).

Background: In Central America, Bothrops asper venom(BaV) is responsible for almost all snakebites. In untreatedcases, local necrosis frequently occurs and may requireamputation. Additionally, many victims of Bothrops aspersnakebite suffer risk of death due to sequels and damageto tissues. Currently, the treatment used for snake biteaccident is the antivenom therapy (AV). However, AV hasbeen shown ineffective in the neutralization of local effectcaused by bothropic venom.

Objective: In the present study, a comprehensive anal-ysis of the effect of red and infrared LED treatment on theoutcome of BaV-induced myonecrosis and inflammationwas performed.

Methods: Male Swiss mice, was used. Edema wasevaluated by pletismograph in different time point: 15, 30min, 1, 3 and 6 h after the injection of 2.5 mg/paw of BaV orsaline (control). Leukocyte influx in the peritoneal cavitywas determined by total and differential cell counts at 6 hafter the injection of 2.5 mg/cavity of B. asper venom.Myonecrosis was induced by injection of 50 mg of B. asper inthe right gastrocnemius muscle and was evaluated 24 hafter venom injection. The myotoxic activity was deter-mined by creatine-quinase assay at 3 h in serum and 24 h inmuscle. Mice had received irradiation of Infrared LED (120mW, 945 nm, 38 s) and red LED (110 mW, 635 nm, 41 s)applied immediately, 1 and 2 h after venom injection.

Results: Our results showed that red LED and InfraredLED treatments had reduced the edema formation inplantar by 70% e 52% and in gastrocnemius muscles by 61%and 86%, respectively. Both LED used had significantlyreduced neutrophils migration 6 h after the venom injec-tion. Also, results showed that Infrared LED and red LEDtreatment significantly reduced serum CK levels by 84% and70%, respectively. In addition, LED treatment causeda significant increment in muscle CK contents in bothwavelengths used, after the venom injection. The histologyshowed an improvement, at least in part, of myonecrosis,corroborating the biochemistry finding.

Discussion: LED therapy significantly reduces theinflammation and myonecrosis caused by BAV in both 635nm and 945 nm wavelengths.

Conclusion: Phototherapy should be considered asa potentially therapeutic approach for treatment of thelocal effects of Bothrops species.

Keywords: myonecrosis, inflammation, phototherapy, Bothrops asper10.1016/j.toxicon.2012.04.213

213. Evaluation of Cabenegrin on the Enzymatic Activityand Structure of Basic sPLA2 of Crotalus durissusterrificus Venom

Veronica C.G. Soares 1, Daniel Bristot 1, Camila L. Pires 1,Rafael M. Ximenes 2, H.S.A. Monteiro 2,Daniela de O. Toyama 3, Marcos H. Toyama 1

1General Biology, UNESP - Campus Experimental do Litoral Paulista, SãoVicente, SP Brazil2 Pharmacology and Physiology Department, Faculdade de Medicina -Universidade Federal do CE Brazil3CCBS, Universidade Presbiteriana Mackenzie, SP BrazilE-mail address: [email protected] (M.H. Toyama).





Background: Pterocarpans are naturally occurring plantproducts carrying a cis-fused benzofuranyl-benzopyranring system. Many of them are isoflavonoids possessinghigh antifungal and antibacterial activity and these naturalproducts cabenegrin A-1 (CA-I) are active components ofa Brazilian folk medicine used against snake venoms. Thesepotent antidotes have been isolated by Nakanishi andcoworkers from the aqueous alcoholic extract of the root ofa South American plant called “Cabeca de Negra”. On theother hand constitute a basic sPLA2 Fractions moreimprotante venom of Crotalus durissus ssp with no knowneffects of CA-I or CA-II on the structure and enzymaticactivity of basic sPLA2.

Methods: Reverse HPLC analysis coupled to circular,UV-Vis and fluorescence detector in tanden, ELISA platereader for enzymatic activity.

Results: In this article we investigated the effect ofthese compounds on the structures of native basic sPLA2from the Bothrops jararacussu and examine the extensionof the previous incubation of sPLA2 with CA-I and its iso-form CA-II on the enzymatic activity of sPLA2, that wereperformed on the ELISA reader. Both CA-I and CA-II shiftthe retention time of the isolated sPLA2 in 1.03 and 1.1minutes in comparisonwith native sPLA2. Both compoundCA-1 and CA-2 decrease the Vmax of the native sPLA2 fromVo¼A425nm (0.556 � 0.03) for 0.34 � 0.02 and 0.28 �0.03, respectively. The analysis performed in wavelengthrange 260-320 nm did not show a significant shifts ofbetween native sPLA2 to sPLA2: CA I and native sPLA2 tosPLA2: CA II and the results from the analysis performed inthe wavelength range 220–260 nm did not allowed toobserve a significant increasing of CD profiles of sPLA2: CAI to native sPLA2 and sPLA2: CA II to sPLA2. By other side,the monitoring of CD spectra in thewavelength range from220 to 260nm showed clear diminishing of the apha-helixand betha strand after incubation of sPLA2 with CA-I orCA-II. The global analysis of the results from the fluores-cence scanning assay suggest that increasing of the fluo-rescence properties of CA I: sPLA2 and CA II: sPLA2probably involve the summation of the fluorescencespectra of sPLA2 and CA I or CA II residues coupled to sPLA2and this contributed for the CD spectroscopic analysis ofsPLA2:CAI and sPLA2:CAII.

Discussion and Conclusion: Neither CA-I and CA-II didnot induced drastic protein unfolding, but stronglydecrease the enzymatic activity of native sPLA2.

Keywords: isoflavone, pterocarpans, basic sPLA2, circular dichroism, fluo-rescence spectroscopy10.1016/j.toxicon.2012.04.214

214. Evaluation of the Antiophidic Activity of Lapacholand Synthetic Analogues

Marcelo A. Strauch 1, Marcelo A. Tomaz 1,Marcos M. Machado 1, Jhonatha M.T. Cruz 1, Bruno L. Cons 1,Alcides J.M. da Silva 2, Paulo R.R. Costa 2, Paulo A. Melo 1

1 Program of Pharmacology and Medicinal Chemistry, Federal University ofRio de Janeiro - Brazil2Natural Products Research Unit, Federal University of Rio de Janeiro - BrazilE-mail address: [email protected] (M.A. Tomaz).

Background: Serotherapy against snakebite wasdiscovered more than one hundred years ago, but theantivenin are not available all over Brazil nor in some partsof the world. The use of plants in folk medicine is commonmainly in the Brazilian Amazon area. We have investigatedthe antiophydic activity of Lapachol (Fig.1-1) isolated fromTabebuia impetiginosa and analogues (Fig.1) synthesized byusing Suzuki-Miyaura coupling methodology, in differentexperimental protocols against Bothrops atrox and Bothropsjararaca venoms.

Methods: We investigated the effects of the naturalproduct and its analogues on venom phospholipase, colla-genase and proteolytic activity. The antiproteolytic activitywas assessed by using azocasein as substrate (Arch of Bio-chem and Biophy, 315:188, 1978) and the phospholipaseactivity by a modified turbidimetric method (Biochm Bio-phys Acta, 554:98, 1965) using a suspension of chicken eggyolk as substrate. The collagenase activity was assessed bythe modified method of Chavira et al. (1984). The hemor-rhagic lesions were induced in mice by an intradermicinjection of B. jararaca venom and quantified as previouslydescribed (Melo et al., 1994).

Results: The lapachol and synthetic analogues inhibitedthe proteolytic and collagenase activity of Bothrops atroxvenom, and also showed anti-hemorrhagic activity in vivoagainst the venom of Bothrops jararaca.

Conclusions: Our studies indicate that Lapachol andsynthetic analogues present relevant inhibition of collage-nase and proteolytic activities of Bothrops atrox venom, butdo not present Phospholipase activity (data not shown),and also inhibited hemorrhagic activity of Bothrops jararacavenom.

Financial Support: CAPES, CNPq, PRONEX and FAPERJ

Keywords: Lapachol, Bothrops atrox , Bothrops jararaca.10.1016/j.toxicon.2012.04.215

215. Ability of Polyanions to Antagonize the CardiotoxicEffect of the Bothrops jararacussu Venom

Himar D. Ricardo, Vinicius V. Martins,Marcos Monteiro-Machado, Marcelo A. Strauch,Marcelo A. Tomaz, Matheus T. Henriques,Bruno Lemos Cons, Jhonatha Mota-Texeira,F.S. Fernanda Siqueira-Lece, Paulo A. MeloLaboratório de Farmacologia das Toxinas, ICB, CCS, UFRJ, 21941-590, Rio deJaneiro, RJ, BrazilE-mail address: [email protected] (P.A. Melo).

Background: Previous studies have showed that B. jar-aracussu venom has myotoxic and cardiotoxic effects andthe myotoxicity is antagonized by heparin. In this study weinvestigated the in vitro cardiotoxic activity of B. jarar-acussu crude venom and the antivenom effects of theheparin and dextran on isolated rat hearts.

Methods: We assessed the venom effects in Wistar ratisolated hearts perfused with an appropriated nutritionalsolution by using the modified Langendorff preparation.We analyzed the tension developed, the electrocardio-graphic records (EKG), the damaged area and the CreatineKinase (CK) activity in the perfusate. The preparation was




perfused under control conditions and after 15 minutes ofstabilization we added the crude B. jararacussu venom (2.5-10 mg/mL) or the venom associated to the polyanions,heparin or dextran sulfate (10-300 mg/ml). At the end of theexperiments, hearts were gently sliced and exposed to 1%triphenyl tetrazolium chloride (TTC) to assess damagedareas (Am Heart J, 593: 101, 1981). Electrical and contractileproperties were analyzed by the WINDAQ program.

Results: The crude venom induced a progressive nega-tive inotropic effect (10 mg/mL) decreasing to 0% the hearttension after 15 min, increasing perfusion press, PRinterval, decreasing QRS amplitude, with changes on theEKG waves, as well increased the rate of CK release in theperfusate. The addition of heparin 30; 100; 300 mg/ml anddextran 10; 30; 100 decreased in concentration-dependentway the venom cardiotoxic effect in the heart tensionreaching 100% of the inhibition with 300 mg/ml (heparin)and 100 mg/ml (dextran), perfusion press and EKG waveschanges. The analysis with TTC showed the damaged areaas well as the protective effect of the polyanions.

Discussion: The venom induced a significant depressionof the cardiac tension, which was antagonized by heparinor dextran sulfate at different concentrations and thefunctional data were confirmed by the TTC stained tissueand the CK release.

Conclusions: This data confirm that polyanionsprotect cardiac muscle fibers from the cytotoxic effect ofB. Jararacussu crude venom.

Financial Support by: CAPES, CNPq, PRONEX e FAPERJ

Keywords: snake venom, cardiotoxic effects, antagonism, polyanions10.1016/j.toxicon.2012.04.216

216. Experimental, Immunochemical Reactivity andNeutralizing Capacity of Rhinocerophis (Bothrops)alternatus Antivenoms, from Distinct GeographicalSources

L.C. Lanari 1, A. Olvera 2, V. Costa de Oliveira 1,3,R.D. Laskowicz 1, P.I. Regner 1,3, F. Olvera 2, R.P. Stock 2,A. Alagón 2, A.R. de Roodt 1,31Área Investigación y Desarrollo/Serpentario, Instituto Nacional deProducción de Biológicos, Administración Nacional de Laboratorios eInstitutos de Salud, Ministerio de Salud de la Nación, Argentina2 Instituto de Biotecnología de la Universidad Autónoma de México,Cuernavaca, Morelos, México3 Laboratorio de Toxinopatología, Centro de Patología Experimental yAplicada, Facultad de Medicina, Universidad de Buenos Aires, Uriburu 950,5�Piso, Lab.555 (1427) Buenos Aires, ArgentinaE-mail address: [email protected] (A.R. de Roodt).

Background: Biochemical and toxicological variation insnake venoms can be observed within the same species.We observed significant geographical variation in toxicityin the snake Bothrops (Rhinocerophis) alternatus fromArgentina, the biggest viper in the South of South America.In addition, variation was also detected in the neutralizingcapacity of therapeutic antivenoms for venom from thesesnakes from different regions, and even in snakes from thesame region. In view of these results, we produced exper-imental antisera against pools of venoms of Bothropsalternatus from different regions of the country.

Materials and Methods: Venoms used were fromBaradero, San Nicolás, Olavarría and Dock Sud (Province ofBuenos Aires), Concordia and Gualeguay (Province of EntreRíos), the provinces of Córdoba, Misiones and Corrientes. APool of venom was generated by mixing equal amounts ofeach of these regional venoms. Rabbits were immunizedwith the different pools of venom and the general pool andwere bled after a fixed immune response was attained(dilution 1/10 by immunodiffusion). IgG was purified bycaprylic acid fractionation, its purity tested by SDS-PAGEand the amount of protein adjusted. The immunochemicalcross reactivity was evaluated by direct ELISA, competitiveELISA assays and, additionally, the avidity of antibodies wasevaluated. Neutralizing capacity on hemorrhagic, coagulantand thrombin-like activities of the antivenoms weremeasured.

Results: An important cross-reactivity, without signifi-cant differences regarding the specificity of the immuno-gens, was observed in the majority of the assays. Thecompetitive assays showed similar results and the avidityexperiments in presence urea (2 M) did not reveal differ-ences in the avidity of the homologous or heterologousvenoms and antivenoms. Neutralization of hemorrhagewas very variable, and neutralization of coagulant activities(on plasma and on fibrinogen) was evident in all cases.Some antivenoms neutralized toxic activities stronglypresent in heterologous venoms but absent or lower in thevenom used as immunogen (i.e. thrombin like activity).

Discussion: Although some differences that could beattributed to geographic isolation of snake populationswere observed, cross-neutralization of venoms of differentregions was widespread. Despite the different toxicpotencies of venoms from different regions, antivenomsfrom snakes of a given region exhibited high immunolog-ical cross reactivity and neutralization on the venoms fromsnakes of very distant and different regions. These findingscould be used to improve the generation of pools for anti-venom production.

Keywords: Rhinocerophis alternatus, Bothrops alternatus, venom variability,cross reactivity, immunology, antivenom10.1016/j.toxicon.2012.04.217

217. IgY Antibodies Anti-Crotalus durissus cumanensisVenom: Purification and Neutralization Efficacy

Montero P. Yuyibeth, Alvarez O. Aurora, Jimenez E. Eucarys,Zerpa Noraida, Malave CaridadInstituto de Estudios Avanzados (IDEA). Centro de Biociencias.Miranda,VenezuelaE-mail address: [email protected] (M.P. Yuyibeth).

Review: The Venezuelan rattlesnake Crotalus durissuscumanensis (Crotalus d.c) is broadly extended across thecountry, causing more than 33% snakebite cases inVenezuela. Avian antibodies (IgY) isolated from chicken eggyolk represent a newalternative to be applied as antivenomtheraphies. In this work we produce IgY antibodies againstCrotalus d.c venom pooled from different Venezuelanregions and evaluate its neutralizing capacity both "invitro" and "in vivo".




Methods: The anti-snake venom antibodies were puri-fied by precipitation techniques with polyethylene glycoland evaluated byMABA, an indirect ELISA, andwestern blotassays. The neutralization of lethality was evaluated bypreincubation of venom together with antivenom prior totesting.

Results: The specificity of IgY antibodies was demon-strated by a dose-dependent inhibition in western blotassay when antibodies pre-absorbed with the venom didnot recognize the venom proteins from Crotalus d.c. Theantivenom was effective in neutralizing 4LD50 doses ofCrotalus d.c. venom in vivo (72 mg of IgY neutralized 1.17mg of Crotalus d.c. venom).

Conclusion: Our results support the future use of aviananti-snake Crotalus d.c. venom as an alternative toconventional equine antivenom in our country.

Keywords: anti-venom, IgY antibodies, snake, neutralization10.1016/j.toxicon.2012.04.218

218. Purification of a Metalloprotease from Najanigricollis Venom and Production of PolyclonalAntibodies

Andrew J. Nok, Binta G. KurfiDept of Biochemistry, Ahmadu Bello University Zaria. Dept of Biochemistry,Bayero University Kano NigeriaE-mail address: [email protected] (B.G. Kurfi).

Background: The snakes particularly responsible forserious medical emergencies in Northen Nigeria includeNaja nigricollis. Bites are common in remote area whereaccessibility to a hospital is unlikely. There is a need tobetter understand the pathophysiology of venom compo-nents and to explore alternative treatments.

Methods: A metalloprotease was isolated from thevenom of Naja nigricollis and purified to apparent electro-phoretic homogeneity by successive chromatography onSephadex G.75 and DEAE-Cellulose columns. Polyclonalantibodies against the metalloenzyme were recoveredfrom mice vaccinated with purified enzyme.

Results: The purified enzymewas optimally active at pH6.2 andwas completely inactivated at pH 4.0. The activity ofthe enzyme increased progressively with temperature to anoptimum of 40oC. The Arrhenius plot from the temperaturedata gave activation energy of 13.40 kJ/mole. The molecularweight of the enzyme determined by size exclusion chro-matography on Sephadex G.75 was 25 kDa. Also the puri-fied enzyme migrated as single 25 kDa band on SDSpolyacrylamide gel. Kinetic analysis of the enzyme frominitial velocity data gave KM and Vmax values of 5.4 mM and10.6 mmoles/min, respectively when casein was used asa substrate. The enzyme was strongly inhibited by EDTAdose dependently. Delineation of inhibition type revealednon-competitive patterns. The Met-p polyclonal antibodiesagglutinated the purified enzyme even at low concentra-tions. The polyclonal antibodies also inhibited the hemor-rhagic activity of the crude enzyme.

Conclusions: Metaloprotease from the venom of Najanigricolis is a small sized monomeric protein of 25kDa.

Polyclonal antibodies raised against the enzyme havethe potential of ameliorating snake envenomation bydiminishing the agglutination time of treated blood. Thepolyclonal antibody inhibition of Cysteine Protease fromPlasmodium berghei could imply possible homology in theactive sites of the enzymes that can be exploited forsystemic drug design.

Keywords: metalloprotease, Naja nicricollis, venom, polyclonal antibodies,Plasmodium berghei10.1016/j.toxicon.2012.04.219

219. Functional Characterization of a RecombinantMyotoxin Inhibitor from Bothrops alternatus SnakePlasma

Norival A. Santos-Filho 1, Danilo L. Menaldo 1,Marco A. Sartim 1, Adélia C.O. Cintra 1, Johara B. França 2,Ludier K. Santos-Silva 3, Flavio H. Silva 3, Eliane C. Arantes 2,Suely V. Sampaio 1

1Departamento de Análises Clínicas, Toxicológicas e Bromatológicas,Faculdade de Ciências Farmacêuticas de Ribeirão Preto, Universidade de SãoPaulo, Ribeirão Preto, Brazil2Departamento de Física e Química, Faculdade de Ciências Farmacêuticas deRibeirão Preto, Universidade de São Paulo, Ribeirão Preto, Brazil3Departamento de Genética e Evolução, Universidade Federal de São Carlos,São Carlos, BrazilE-mail address: [email protected] (N.A. Santos-Filho).

Background: Myonecrosis is an important medicalcomplication resulting from snakes bites and, in severecases, the local myonecrosis may lead to drastic conse-quences, such as permanent tissue loss, disability, or limbamputation. The venomous and non-venomous snakeshave in their blood serum, proteins which inhibit myo-toxins, called PLIS.

Methods: The ability of the recombinant inhibitor(rBaltMIP) to inhibit the enzymatic activity of PLA2s Asp49(BthTX-II, BthA-I-PLA2, PrTX-III and CB) was evaluated byindirect radial hemolysis method, performed on plates. Themyotoxic activity of Lys49 and Asp49 myotoxins (BthTX-I,BthTX-II, PrTX-I and PrTX-III) was carried out by measuringof the Creatine Kinase (CK) levels in mice plasma. Either forphospholipase activity, and for the coagulant activity, PLA2sand PLA2-like were previously incubated for 30 min aloneor with rBaltMIP in different molar ratios.

Results: It was possible to observe a slight reduction inthe phospholipase activity of PLA2s, with maximuminhibitory values of approximately 40% for enzymes BthTX-II and PrTX-III. The recombinant inhibitor was effective inreducing the myotoxicity of myotoxins tested, especiallyBthTX-I and PrTX-I, both Lys49 PLA2-like. However, it maybe noted that despite a higher efficiency of inhibitionagainst the Lys49 PLA2-like, rBaltMIP showed a significantinhibitory activity against the Asp49 PLA2s myotoxins(approximately 50%). It was also performed the inhibitionof the activity of different myotoxic PLA2s myotoxinswithout the pre-incubation with the inhibitor. rBaltMIPshowed considerable ability to inhibit the myotoxic activityof different myotoxins (50%), mainly for Lys49 PLA2-likemyotoxins. Was also tested the viability of serotherapy




complementation by rBaltMIP. In this activity, it wasobserved that the snake antivenom for veterinary use wascapable of neutralizing the myotoxic activity of Bothropsjararacussu venom and BthTX-I, moreover, the inhibitorrBaltMIP was more effective to neutralize the myotoxicityof these compounds compared to the antiserum tested.Therefore, when the antiserum was supplemented withrBaltMIP was possible to observe a complementation of itsinhibition activity, suggesting a possible commercialapplication for this inhibitor.

Discussion and Conclusions: It is known that myo-toxins has a low immunogenicity, which means that anti-venom is not fully effective for those enzymes. rBaltMIPshowed high potential for complementation of snakeantivenom, however, future studies should be performed.

Keywords: rBaltMIP, PLI, myotoxin inhibitor, myotoxic activity10.1016/j.toxicon.2012.04.220

220. Evaluation of Extracts and Partitions from AerialParts of Baccharis microdonta on Enzymatic Activity,Pro-Inflammatory and Myotoxic Activities Induced bySecretory Phospholipase A2 from Bothrops jararacussu

Veronica C.G. Soares 1, Daniel Bristot 1, Camila L. Pires 1,Marcos H. Toyama 1, Paulete Romoff 2, Marcelo J. Pena 2,Oriana A. Favero 3, Daniela de O. Toyama 3

1General Biology, UNESP - Campus Experimental do Litoral Paulista,SP-Brazil2Centro de Ciências e Humaniidades - Universidade PresbiterianaMackenzie, SP-Brazil3Centro de Ciências Biológicas e da Saúde - Universidade PresbiterianaMackenzie, SP-BrazilE-mail address: [email protected] (D.deO. Toyama).

Background: Several species of Baccharis have beenextensively used in folk medicine for the treatment orprevention of anemia, diabetes and stomach, and otherinflammatory diseases. As the inflammation involves themobilization of arachidonic acid by phospholipase A2, thisproject used the sPLA2 purified from the Bothrops jarar-acussu as molecular target for the initial studies for char-acterize the anti-phospholipasic A2 of the methanol extractand its partitions in hexane, dichloromethane, ethyl acetateand Butane -1-ol.

Methods: For purification of sPLA2 from Bothrops jar-acussu used a combination of ion exchange and reversephase HPLC, for obtantion methanol extraction and theirrespective partitions. The enzymatic activity was doneusing a NOB and all experiments were done on the ELISAplate reader. The Baccharis microdonta aerial parts wereprocessed for preparation of methanolic was partitionedwith hexane, butane-1-ol, dichloromethane and ethylacetate partitions.

Results: The inhibition enzymatic activity of phospho-lipase A2 was observed only for the partition butane-1-olwhile the methanolic extract and the other partitionsshowed a little or absent. The paw edema test performed inmice were performed with native sPLA 2 from Bothropsjararacussu, sPLA2 previously treated with the extract andits partitions, which they have also been previously

injected into the animals 30 minutes before the adminis-tration of native sPLA2. Under these conditions, weobserved that the methanol extract and dichloromethanepartition abolished the edematogenic effect of sPLA2 ofnative Bothrops jaracussu PLA2. The partition in Ethylacetate was injected 30 minutes before abolished theedema induced by native sPLA2. The ethyl acetate partitionpreviously injected 30 minutes before or previously incu-bated with the native sPLA2 abolished the myotoxic effectsof sPLA2. These results suggest that the methanol extractand dichloromethane partitions showed a promising anti-inflammatory activity but did not directly inhibit phos-pholipase A2. The real-time PCR results showed a decreasein synthesis of COX-1, so that the partition dichloro-methane and methanol extract can inhibit the inflamma-tory activity of sPLA 2 via inhibition of COX-1.

Discussion: The previous result analysis showed thatBaccharis microdonta present some bioactive compoundthat inhibited the sPLA2 enzymatic activity that seen onlyfound in the butane-1-ol partition that in general allowedthe purification of glycosiled flavonoids and tannins thatdid not extracted by other partition.

Keywords: Baccharis microdonta, phospholipase A2, Bothrops jaracussu,COX10.1016/j.toxicon.2012.04.221

221. The Interaction of the Antitoxin DM43 with a SnakeVenom Metalloproteinase Analyzed by MassSpectrometry and Surface Plasmon Resonance

Guilherme D. Brand 1, Rune Salbo 2,Thomas J.D. Jørgensen 3, Carlos Bloch Jr.1,Elisabetta B. Erba 4, Carol V. Robinson 5, Isabelle Tanjoni 6,Ana M. Moura-da-Silva 6, Peter Roepstorff 3,Gilberto B. Domont 7,9,10, Jonas Perales 8,9,10,Richard H. Valente 8,9,10, Ana G.C. Neves-Ferreira 8,9,10

1 Laboratory of Mass Spectrometry, Embrapa-Recursos Genéticos eBiotecnologia, Brazil2Diabetes Protein Engineering, Novo Nordisk A/S, Denmark3Department of Biochemistry and Molecular Biology, University of SouthernDenmark, Denmark4 Laboratory of Organic Chemistry, ETH Zürich, Switzerland5 Physical and Theoretical Chemistry Laboratory, Department of Chemistry,University of Oxford, United Kingdom6 Laboratory of Immunopathology, Butantan Institute, Brazil7 Laboratory of Protein Chemistry, Chemistry Institute, Federal University ofRio de Janeiro, Brazil8 Laboratory of Toxinology, Oswaldo Cruz Institute, Fiocruz, Brazil9Rio de Janeiro Proteomic Network/FAPERJ, Brazil10 INCTTOX /CNPq, BrazilE-mail address: [email protected] (R.H. Valente).

Background: Natural inhibitors of snake toxins arepromising molecules that can contribute to the rationaldevelopment of new ancillary snakebite envenomationtherapies. DM43 is a circulating dimeric antitoxin isolatedfrom Didelphis aurita, a South American marsupial natu-rally immune to snake envenomation. This endogenousinhibitor binds non-covalently to jararhagin, the mainhemorrhagic metalloproteinase from Bothrops jararacasnake venom, and efficiently neutralizes its toxicity. Theaim of this study was to apply mass spectrometry and




surface plasmon resonance to improve the molecularcharacterization of this heterocomplex.

Methods: Mass spectrometry (nanoESI-Q/TOF MS) wasused to analyze jararhagin, DM43 and the stoichiometry oftheir toxin-antitoxin complex; moreover, the quaternarystructure of DM43 was assessed by this methodology. Therate and equilibrium constants of the interaction weredetermined by surface plasmon resonance. The toxin wascaptured on a sensor chip derivatized with the anti-jar-arhaginmonoclonal antibodyMAJar 2 and the sensorgramsobtained after successive injections of DM43 in a concen-tration series were globally fitted to a simple bimolecularinteraction.

Results and Discussion: The stoichiometry of theinteraction was confirmed by MS; from native solutionconditions, the complex showed a molecular mass of w 94kDa, indicating that one molecule of jararhagin (50 kDa)interacts with one monomer of DM43 (43 kDa). Althoughreadily observed in solution, the dimeric structure of theinhibitor was barely preserved in the gas phase. This resultsuggests that, in contrast to the toxin-antitoxin complex,hydrophobic interactions are the primary driving force forthe inhibitor dimerization. Regarding the real-time inter-action analysis, the following kinetic rates, for the DM43/jararhagin interaction, were obtained: ka ¼ 3.54 � 0.03 �104 M-1s-1 and kd ¼ 1.16 � 0.07 � 10-5 s-1, resulting in anequilibrium dissociation constant (KD) of 0.33 � 0.06 nM.

Conclusions: The binding stoichiometry data deter-mined by MS unequivocally show that one monomer ofDM43 binds to one jararhaginmolecule. Moreover, we haveshown that DM43 dimers, which are rather stable in solu-tion, are not preserved in the gas phase corroborating thatits dimerization interfaces may be due to interactionbetween hydrophobic residues, a theoretical predictionbased on PATCHES analysis. Finally, the kinetic character-ization of this interaction indicates that DM43 binds to thereferred target toxin with high affinity and may constitutea suitable template for the rational development of novelhighly specific therapeutic agents to modulate the activityof SVMPs and their homologues.

Keywords: metalloproteinase, metalloproteinase inhibitor, snake venom,toxin, antitoxin, mass spectrometry, surface plasmon resonance10.1016/j.toxicon.2012.04.222

222. Molecular Mechanisms Involved in PGE2 ReleaseInduced by the Snake Venom Metalloproteinase BaP1 inSynoviocytes

Mariana Viana 1, Catarina Teixeira 1, Elbio Leiguez 1,José M. Gutiérrez 2, Alexandra Rucavado 2,Cristina M. Fernandes 11Unity of Inflammation, Laboratory of Pharmacology, Butantan Institute, SaoPaulo, Brazil2Clodomiro Picado Institute, University of Costa Rica, San José, CRE-mail address: [email protected] (C.M. Fernandes).

Background: Snake venommetalloproteinases (SVMPs)and Matrix metalloproteinases (MMPs) share commondomain organization and exhibit identical Zn-bindingmotif. Studies on SVMPs may thus provide insights into the

functions of MMPs. Levels of these enzymes are increasedin inflamed articular joints. Articular synovial fibroblasts(B type) are the main cells involved in production andrelease of inflammatory mediators during joint inflamma-tory processes, being PGE2 a major mediator. BaP1 is a 22.7kDa SVMP from B. asper snake venom and contains only themetalloproteinase domain. This enzyme displays potentinflammatory activities both in vivo and in vitro experi-mental models. In this study we investigated the effects ofthis SVMP on isolated synoviocytes focusing on the releaseof prostaglandin E2 (PGE2) and the molecular mechanismsinvolved in this effect.

Methods: B type synoviocytes isolated from rat kneejoints synovial membranes (CEUIAB 576/09) were used.Levels of PGE2 were measured by enzyme immunoassayand protein expression of the PGE2 receptor EP4 wasdetermined by Western blotting. Participation of bothNF-kB and EP4 receptor in the release of PGE2 and COX-2protein expression induced by BaP1 was evaluated in cellspretreated with the inhibitors SN50 (50 mg/mL) andAH23848 (30 mM), respectively.

Results: BaP1 induced release of PGE2 from B typesynoviocytes after 1, 3, 6 and 12 h, but not 30 min incu-bation, in comparison with control cells incubated withRPMI alone. BaP1 induced EP4 protein expression (52 and65 kDa) by synoviocytes (30min – 6h). Moreover, inhibitionof NF-kB or EP4 receptor significantly decreased BaP1-induced PGE2 release and COX-2 protein expression.

Discussion: These data indicate the ability of BaP1 toinduce biosynthesis of PGE2 and COX-2 expression in iso-lated B type synoviocytes. These effects aremediated byNF-kB pathway. Moreover, data demonstrated that EP4receptor regulates BaP1-induced PGE2 production and COX-2 expression through a positive feedback loop, and stronglysuggest that this subtype of PGE2 receptor contributes foramplification of BaP1-induced release of PGE2.

Conclusion: BaP1 can directly stimulate synoviocytesfor synthesis of PGE2 and expression of both COX-2 enzymeand EP4 receptor. These findings suggest novelmechanismsof action displayed by metalloproteinases in synoviocytes.

Financial support: FAPESP; CNPq

Keywords: metalloproteinase, prostaglandin E2, synoviocyte10.1016/j.toxicon.2012.04.223

223. TLR2 and MyD88 Signaling are Required forEfficient Response of Macrophages to MT-IIIa Phospholipase A2 (PLA2) from Bothrops asper SnakeVenom

Elbio Leiguez 1, Karina C. Giannotti 1, Vanessa Moreira 1,Márcio H. Matsubara 1, Bruno Lomonte 2,Catarina F.P. Teixeira 1

1Butantan Institute, Laboratory of Pharmacology, Sao Paulo, Brazil2Clodomiro Picado Institute, University of Costa Rica, San José, CRE-mail address: [email protected] (C.F.P. Teixeira).

Background: Toll-like receptors (TLRs) are majorcomponents of the innate immune system and primarysensors for noxious stimuli. Upon activation these




receptors initiate an immediate inflammatory responsethrough the myeloid differentiation factor 8 (MyD88)adaptor protein signal transduction. TLRs are highlyexpressed on macrophages, which are key cells of inflam-mation. MT-III is a PLA2 with myotoxic and inflammatoryactivities. This enzyme induces inflammatory events andactivates macrophages functions, including release ofinflammatory mediators, phagocytosis and lipid body (LB)formation. However, the role of the TLR system in theinflammatory actions of venom PLA2s is unknown. In thisstudy we investigated the role of TLR2 and MyD88 adaptorprotein in the release of eicosanoids (PGE2, PGD2,TXA2, LTB4), cyclooxygenase-2 (COX-2) expression and LBformation induced by MT-III in macrophages.

Methods: Peritoneal macrophages were obtained fromC57BL/6 wild type (WT), TLR2-/- and MyD88-/- male miceand stimulated with MT-III (0.4 mM) or RPMI (control) for 6h. PGE2, PGD2, TXA2 and LTB4 levels were quantified by EIAand COX-2 protein expression evaluated byW. blotting. LBswere stained with osmium tetroxide (1%) and countedunder phase contrast microscopy.

Results: Stimulation ofWTmacrophages byMT-III causeda marked release of PGE2, PGD2, TXA2 and LTB4 and increasein numbers of LBs in comparisonwith respective controls. InMT-III-stimulated TLR2-/- macrophages, formation of LBs andrelease of PGE2, LTB4 and PGD2, but not TXA2were abrogated.In MyD88-/- macrophages, release of PGE2 and TXA2 inducedby MT-III was not observed in comparison with WT macro-phages and the ability ofMT-III to induce release of both LTB4and PGD2 was maintained in these MyD88-/- cells. In addi-tion, the ability of MT-III to induce COX-2 protein expressionseen in WT macrophages was significantly reduced in bothTLR2-/- and MyD88-/- macrophages.

Discussion: Obtained data indicate the involvement ofTLR2 and MyD88 to the innate immune response to MT-IIIthrough activation of macrophages since the absence ofthese pathways resulted in deficiency in release ofinflammatory mediators and LB formation by these cells.Both TLR2 and MyD88 signaling are crucial to PGE2biosynthesis, COX-2 expression and LB formation inducedby MT-III. Moreover, TLR2 is relevant to LTB4 and PGD2biosynthesis induced by MT-III, whereas MyD88 signaltransduction is essential to TXA2 biosynthesis.

Conclusion: TLR2 and MyD88 signaling are relevant inactivation of defense functions of macrophages by thesnake venom PLA2 MT-III.

Financial support: INCTTOX, CNPq, FAPESP

Keywords: snake venom phospholipase A2, toll-like receptor,macrophages10.1016/j.toxicon.2012.04.224

224. Snake Venomics of Crotalus tigris. EvolutionaryClues for Generating a Pan-Specific Antivenom AgainstCrotalid Type II Venoms

Juan J. Calvete 1, Alicia Pérez 1, Bruno Lomonte 2,Elda E. Sánchez 3, Libia Sanz 1

1 Instituto de Biomedicina de Valencia CSIC, Valencia, Spain

2 Instituto ClodomiroPicado, Facultad de Microbiología, Universidad de CostaRica, San José, Costa Rica3National Natural Toxins Research Center, Deparment of Chemistry, TexasA&M University-Kingsville, Kingsville, Texas, United States

E-mail address: [email protected] (L. Sanz).

Background: The tiger rattlesnake, Crotalus tigris, isa medium-sized ground-dwelling dessert pitviper, thatambushes much of its prey but also active foragessmall rodents and lizards, juveniles relying heavily onlizards and adults depending more on rodents. In addi-tion, this small rattlesnake has been known to eat fairlylarge prey, including kangaroo rats, packrats, and evenspiny lizards. This is based upon its venom's highlethality, rated the highest of all rattlesnake venoms(LD50 value for mice is 0.07 mg/kg intraperitoneal, 0.056mg/kg intravenous, and 0.21 mg/kg subcutaneous). Thecomparatively low venom yield (6.4-11 mg dried venom)and short 4.0-4.6 mm fangs of C. tigris possibly preventsevere envenoming in adult humans. However, the clin-ical picture could be very more serious if the personbitten was a child or a slight build individual. We reportthe proteomic and antivenomic characterization of Cro-talus tigris venom.

Methods: An experimental antiserum was raised inrabbits by subcutaneous injections of subletal amounts ofa mixture of venoms from C.d. terrificus, Crotalus simus andCrotalus lepidus lepidus. The ability of the experimentalantivenom to effectively immunodeplete proteins from thetype II venoms of C. tigris, Crotalus horridus , Crotalusoreganus helleri, Crotalus scutulatus scutulatus, and Sis-trurus catenatus catenatus indicated the feasibility ofgenerating a pan-American anti-Crotalus type II anti-venom, suggested by the identification of shared evolu-tionary trends among South and North American Crotalusspecies.

Results: This venom exhibits the highest lethality formice among rattlesnakes and the simplest toxin pro-teome reported to date. The venom proteome of C.tigris comprises 7-8 gene products from 6 toxin families;the presynaptic b-neurotoxic heterodimeric PLA2, Mojavetoxin, and two serine proteinases comprise, 66 and 27% ofthe C. tigris toxin arsenal, whereas a VEGF-like protein,a CRISP molecule, a medium-sized disintegrin, and 1-2PIII-SVMPs each represent 0.1-5% of the totalvenom proteome. This toxin profile, in particular, the lowmetalloproteinase content, the high concentration ofMojave toxin subunits and its high toxicity LD50 0.05(i.v)-0.07(i.p) mg/g of mouse body weight, place a C.tigrisvenom in the type II class defined by Mackessy. Thevenom composition really explains the systemicneuro and myotoxic effects observed in envenomatedanimals. In addition, we found that venom lethality of C.tigris and other North American rattlesnake type IIvenoms correlates with the concentration of Mojavetoxin A-subunit.

Keywords: North American rattlesnake, Crotalus tigris, snake venomics,antivenomics.10.1016/j.toxicon.2012.04.225



225. Proteomic Analysis and Pharmacological Activitiesof the Venom of the Moroccan Cobra Naja haje legionis.

Ibtissam Malih 1,2,3, Muhamad Rusdi Ahmad Rusmili 2,Ting Yee Tee 2, Rachid Saile 3, Noreddine Ghalim 1,Iekhsan Othman 2

1Venoms and Toxins Laboratory, Pasteur Institute of Morocco, Casablanca,Morocco2Department of Biomedical Sciences, School of Medicine and HealthSciences, Monash University Sunway Campus, Malaysia3URAC 34, Hassan II University Mohammedia - Casablanca, Faculty ofScience Ben M'sik, MoroccoE-mail address: [email protected] (I. Malih).

Background: In Morocco, envenomation by snake bitesposes a serious problem to public health. The cobra Najahaje legionis is endemic of the country and one of the mostdangerous species known. In this work, we report theproteomic and pharmacological characterizations of bio-logically active proteins from the venom of this species.

Methods: The various proteins in the crude venomwerefractionated and analyzed by a combination of chromato-graphic separations and proteomic analysis techniquessuch as gel filtration, RP-HPLC, 1D electrophoresis, in-geldigestion, tandemmass spectrometry and protein databasesearch. The pharmacological properties of Naja haje legionisvenom were assessed using in vitro preparations usingrodents and chicks.

Results: Our venomic strategy allowed the identificationof 64 proteins and peptides from known database which canclassified into 17 families according to their biological activ-ities. We were able to identify cobra venom factor, L-amino-acid oxidases, acetylcholinesterase, metalloproteinase, dis-integrin, cysteine-rich secretory proteins, nerve growthfactor, phospholipases A2, vespryns, kunitz-type inhibitor,short neurotoxins, long neurotoxins, weak neurotoxins,neurotoxin like proteins, muscarinic toxins, cytotoxins andcardiotoxins. Pharmacological tests showed that Naja hajelegionis venom contained neurotoxic activities inducingirreversible blockage of neuromuscular transmission in bothrodent and chick nerve-muscle preparations. This venomalso exhibit myotoxic and cardiotoxic activities.

Conclusions: These results showed the potency andthe complexityof the variousproteinspresent in thevenomofNajahaje legionis,whichpresent averysimilarpattern toothercobra venoms. The contribution of the different componentsin venom toxicity deserves further investigations.

Keywords: Naja haje legionis, characterization, neurotoxins, proteomics,pharmacology10.1016/j.toxicon.2012.04.226

226. Processing of SVMPs: Detection of SVMP Zymogensand Pro-Domain in Bothrops jararaca Venom andVenom Glands

J.A. Portes-Junior 1, G.S. Magalhães 1, S.S. Sant'Anna 2,M.R. Junqueira 3, N. Yamanouye 4, A.M. Moura-da-Silva 1,G.B. Domont 31 Laboratório de Imunopatologia, Instituto Butantan, Sâo Paulo, SP, Brazil2 Laboratório de Herpetologia, Instituto Butantan, São Paulo, SP, Brazil

3Unidade Proteômica, Instituto de Química, Universidade Federal do Rio deJaneiro, Rio de Janeiro, RJ, Brazil4 Lab. de Farmacologia, Instituto Butantan, São Paulo, SP, BrazilE-mail address: [email protected] (G.B. Domont).

Background: Snake Venom Metalloproteinases(SVMPs) responsible for Bothrops envenoming cause severelocal and systemic complications in humans. They aremultidomain enzymes synthesized as zymogens and theenzyme activation is regulated by hydrolysis of the pro-peptide also known as pro-domain.

Methods: In this work we aim to trace the SVMP pro-cessing route by detecting the zymogenmolecule or cleavedpro-domain in Bothrops jararaca venom glands using anti-pro-domain antibodies and mass spectrometry. The pro-domain was obtained by expression of its cDNA extractedfrom venom glands in pAE vector/E. coli. The recombinantprotein (PD-Jar) was used to immunize mice to obtainpolyclonal antibodies. The presence of pro-domains inzymogenor processed formswas then evaluatedbyWesternBlotting in venom samples and venom gland extractscollected at 0, 4, 7, 10, 14 and 21 days after milking.

Results: The antiserum was able to recognize bands of22 and 47 kDa in venom samples collected 7 and 10 daysafter milking, which contained pro-domain peptidesdetected by MS/MS. In the extracts of venom glands, anti-serum was able to recognize bands of 47 and 76 kDa in allsamples. The reactive bands were extracted by pulldownusing anti-pro-domain antibodies and subjected to char-acterization by mass spectrometry.

Discussion and Conclusions: Our results indicate thatSVMPs are mainly found as zymogens within the venom-secreting cells and processing is very likely to occur in thelumen of the venom gland, in a time-dependent manner.

Supported by: FAPESP, CNPq and CAPES

Keywords: zymogen, SVMPs, Bothrops jararaca, processing10.1016/j.toxicon.2012.04.227

227. Proteopeptidome Determination of Bothropsjararaca Venom: an Innovative Approach in SnakeVenomics

Carolina A. Nicolau 1,3,4, André Teixeira-Ferreira 1,3,4,Paulo C. Carvalho 1,3, Magno Junqueira 2,3,4,Jonas Perales 1,3,4, Ana Gisele -Ferreira C. Neves 1,3,4,Richard H. Valente 1,3,4

1Oswaldo Cruz Foundation, RJ, Brazil2 Federal University of Brasilia, DF, Brazil3 Proteomic Network, RJ, Brazil4 INCTTOX/ CNPq, BrazilE-mail address: [email protected] (C.A. Nicolau).

Background: Aiming for a breakthrough regarding theactual limitations of data volume generated in snake ven-omics analyses, our group implemented the use of iso-electrofocusing technology (OFFGEL) followed by reversed-phase nanochromatography coupled to high resolutionmass spectrometry, in order to simultaneously analyze theproteome and peptidome (PROTEOPEPTIDOME) of Bothropsjararaca venom.





Methodology: Crude venom was fractionated in highresolution mode (24 wells) using 3-10 or 4-7 pH range strips,for peptidomic or proteomic analysis, respectively. Thesamples recovered in solution were further desalted andconcentrated by ZipTip C18 (peptidomics) or digested in solu-tion and desalted by molecular exclusion (7,000 Da cut-off)desalting spin columns (proteomics), followed by mass spec-trometric analysis on an LTQ XL-Orbitrap mass spectrometer.

Results: The proteome analysis identified low abundanceproteins such as ecto-5'-nucleotidase, phosphodiesterase,hyaluronidase, aminopeptidase A and transferrin andallowed the identification of 320 proteins, 36 of themalreadydescribed for B. jararaca venom. These numbers were eighttimes higher than those obtained for this venom analyses byprevious approaches. The peptidome analysis revealed theexistence of a highly complex peptidome, mainly by theidentification of peptides related to other groups of proteins(metalloproteinase, phospholipase A2, serine proteinase, L-amino acid oxidase, vascular endothelial and nerve growthfactors, among others) than the traditionally expected bra-dykinin-potentiating peptide group.

Discussion: The low abundance proteins identified byour proteomic analysis are rarely detected by traditionalproteomic approaches; also, the number of identifiedproteins was significantly higher than previous reports.Regarding the peptidomic approach, our data can initiallybe viewed as a representation of the snake venom degra-domics. On the other hand, we hypothesize that some, if notall, of these peptides represent the venom criptome, that is:the population of peptides [originated by proteolytic cliv-age of their criptein(s)] possessing similar or completelydifferent bioactivity from their precursor molecule. More-over, it is tempting to suggest that they should be chosen asmolecules to be tested against an array of biochemical andpharmacological assays, if one is willing to thrive in thesearch for new bioactive molecules in snake venoms.

Conclusions: The use of OFFGEL coupled to high reso-lution mass spectrometry is an innovative approach to beused for snake venomics studies, in order to contribute toa deeper understanding of the snake biology, the patho-physiology of envenoming, the development of new ther-apeutic drugs and the improvement of antiophidic therapy.

Keywords: OFFGEL, snake venomics, peptidome, proteome, criptome,Bothrops jararaca10.1016/j.toxicon.2012.04.228

228. Venom Variability and Envenoming SeverityOutcomes of the Crotalus scutulatus scutulatus(Mojave Rattlesnake) from Southern Arizona

Daniel J. Massey 1, Juan J. Calvete 2,3, Elda E. Sanchez 4,Libia Sanz 3, Kelvin Richards 1, Ryan Curtis 1, Keith Boesen 1

1Arizona Poison and Drug Information Center, Tucson, AZ, USA2Departamento de Biotecnología, Universidad Politécnica de Valencia,Valencia, Spain3 Laboratorio de Proteinomica Estructural, Instituto de Biomedicina deValencia, CSIC, Valencia, Spain4National Natural Toxins Research Center & Department of Chemistry, TexasA&M University-Kingsville, Kingsville, TX, USAE-mail address: [email protected] (J.J. Calvete).

Background: Snake venoms are well documented ashaving different venom compositions and toxicity basedon taxonomic, geographical locations or intra speciesvariation. The purpose of this study was to determine ifthe geographical differences in venom of the C. s. scutu-latus rattlesnake correlate with increased envenomingseverity outcomes. We chose the counties of Pima andCochise AZ, U.S.A. based on the different venom pheno-types documented within each; Pima type B or AþB,Cochise type A. We hypothesized that Cochise Countywould have more severe envenomations when comparedto Pima County.

Methods: Twenty-one C. s. scutulatus were collectedfrom Arizona and New Mexico U.S.A. Venom proteome ofeach specimenwas analyzed using reverse-phase HPLC andSDS-PAGE. The toxicity of venoms was analyzed usinglethal dose 50 (LD50). Envenoming severity outcomesbetween Pima and Cochise counties were determined byretrospective chart review of the APDIC database betweenthe years of 2002-2009.

Results: Six phenotypes (A-F) were identified basedon three venom protein families; Mojave toxin, snakevenom metalloproteinases PI and PIII (SVMP), andmyotoxin. Venom changed geographically from SVMP-rich to Mojave toxin-rich phenotypes as you move fromsouth central to southeastern Arizona. Phenotypes con-taining myotoxins were only found in the transitionalzone between the SVMP and Mojave toxin phenotypes.Venom samples containing the largest amounts of SVMPor Mojave toxin had the highest and lowest LD50s,respectively. A significant difference was found whencomparing presence of neurotoxic effects (p ¼ 0.001). Nosignificant difference was found when comparingseverity (p ¼ 0.32), number of antivenom vials admin-istered (p ¼ 0.17), days spent in a health care facility (p¼ 0.23) or envenomation per 100,000 population (p ¼0.06). Also noted, there is a 10x and 50x increased riskof death or intubations if envenomated in CochiseCounty.

Discussion: Our results support and extended thefindings of Glenn/Straight. Six venom phenotypes basedon neurotoxic (A), hemorrhagic (B), or myotoxic (M)compositions were identified. Types A, B, AþB aspreviously described, also, AþM, BþM, AþBþM. Despitean increased risk of death or intubations if envenomatedin Cochise County or the differences in venom pheno-types identified between Pima and Cochise counties,no significant difference was found in envenomingoutcomes.

Conclusion: Clinical manifestations may not have beenrepresented accurately based on the limiting nature ofretrospective chart reviews. Following future envenoma-tions on a case-by-case basis may reveal a better correlationwith venom proteomics and clinical envenomationmanifestations.

Keywords: snake venom, geographic venom variability, venomics,mass spectrometry, snake envenoming, Crotalus scutulatus, Mojaverattlesnake10.1016/j.toxicon.2012.04.229



229. Geographic Variation of Venom Proteins andNeurotoxicity in the Southern Pacific Rattlesnake(Crotalus oreganus helleri)

Eric Gren 1, Wayne C. Hodgson 2, Rachelle Kornhauser 2,Carl Person 1, Wayne Kelln 1, William K. Hayes 1

1Department of Earth and Biological Sciences, Loma Linda University, LomaLinda, CA, USA2Monash Venom Group, Department of Pharmacology, Monash University,Victoria, Australia

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

Background: Snake venoms are comprised of incrediblycomplex protein arsenals capable of inducing a wide rangeof physiological effects upon envenomation. Althoughsnakes generally present species-specific venom proteinprofiles, venom proteins can also vary significantly withina species. Intraspecies venomvariation is often seen amonggeographically separate populations and can also occuramong individuals within a population or within individ-uals over time. The Southern Pacific rattlesnake (Crotalusoreganus helleri) has traditionally been characterized ashaving primarily proteolytic venom; however, neurotox-icity has been documented in a few individuals. A previousstudy reported Mojave toxin (MT), a heterodimeric phos-pholipase A2 (PLA2) b-neurotoxin found in the venom ofthe Mohave rattlesnake (C. scutulatus), in the venom ofsome C. o. helleri individuals. Mojave toxin, however, fails toaccount for all C. o. helleri neurotoxicity, as neurotoxicenvenomations have been documented in individualsshown not to possess MT.

Methods: In the present study, 2D SDS-PAGE and liquidchromatography were used to obtain venom proteinprofiles for individual C. o. helleri collected across thespecies’ geographic range. Chick biventer cervicis nerve-muscle preparations were used as non-specific physiolog-ical assays of lyophilized venom samples for neurotoxicactivity. Biologically relevant crude venom doses (5 mg)were also injected into mice. Chromatography peak frac-tions were subjected to trypsin digestion and analyzedusing liquid chromatography–mass spectrometry. Searcheswere performed for amino acid sequence similarity in theSwissProt database.

Results and Discussion: Mass spectrometryconfirmed the presence of MT subunits in several C. o.helleri venom samples, and these samples showedneurotoxic activity in the nerve-muscle preparations.However, some C. o. helleri venoms lacking the MTsubunits also exhibited neurotoxicity in the nerve-muscleassays. Simulated envenomations of mice resulted inimmobilization and death in the following order of mostrapid to slowest: C. o. helleri with MT, C. o. helleri lackingMT, C. s. scutulatus with MT. C. o. helleri venoms expressseveral basic (and some acidic) peptides in the w4-7 kDarange which appear exceptionally abundant (as % totalprotein) and variable compared to other rattlesnake taxa.These small basic peptides may account, at least in part,for the non-MT neurotoxicity and increased overalltoxicity observed in some C. o. helleri. Additional datafrom on-going investigation of these proteins will bepresented. We thank Richard Straight, PhD for his helpful

discussions on geographic variability in C. o. hellerivenoms.

Keywords: Crotalus oreganus helleri, venom variability, snake venomneurotoxicity, snake venomics10.1016/j.toxicon.2012.04.230

230. Determining the Interaction Region Between theAntimyotoxin DM64 and a Snake Venom Myotoxin

S.L.G. Rocha 1, A.G.C. Neves-Ferreira 1, M.R.O. Trugilho 1,R.H. Valente 1, G.B. Domont 2, J. Perales 1

1 Laboratório de Toxinologia, IOC, Fiocruz, RJ, Brazil2 Laboratório de Química de Proteínas, IQ, UFRJ, RJ, BrazilE-mail address: [email protected] (J. Perales).

Review: Snake envenomations represent a publichealth problem in tropical countries. The opossum Didel-phis aurita is resistant to the toxic effects of snake venomsdue to the presence of serum neutralizing factors such asDM43 and DM64. The latter is a glycoprotein able toinhibit the in vivo myotoxicity and the in vitro cytotoxicityof myotoxins I (D49) and II (K49) from the venom ofBothrops asper, without inhibiting the phospholipase A2activity of the first one. Our study aimed to map the regionof interaction between DM64 and myotoxin II fromBothrops asper venom. The hydrolysis of DM64 by Lys-Cgenerated several peptides that were chromatographedthrough a myotoxin/NHS-Sepharose affinity column. ThreeDM64 peptides bound to the column were identified byMS/MS: two located in the third domain and another onein the fifth domain of the inhibitor. Alternatively, DM64was cross-linked to the myotoxin using BS3, followed bytrypsinization of the complex and nLC-LTQ-Orbitrapanalysis. Most observed cross-links occurred betweenresidues K241 (third domain) of DM64 and K15 of myo-toxin II, and also between K452 (fifth domain) of DM64and K60 of myotoxin II. In summary, the interaction ofDM64 with myotoxin II seems to involve the second andfifth domains of the inhibitor, as suggested by twocomplementary methodological approaches. These resultscontribute to the molecular characterization of thisimportant non-covalent complex and open the perspec-tive that DM64 or its peptides may be used in the treat-ment of snake envenomations.

Financial support: Fiocruz, CNPq, Faperj, INCTTOX

Keywords: DM64, myotoxin, cross-linked10.1016/j.toxicon.2012.04.231

231. Second Generation Antivenomics: ComparingImmunoaffinity and Immunodepletion Protocols

Davinia Pla 1, José M. Gutiérrez 2, Juan J. Calvete 1

1 Instituto de Biomedicina de Valencia, CSIC, Valencia, Spain2 Instituto Clodomiro Picado, Facultad de Microbiología, Universidad de CostaRica, San José, Costa RicaE-mail address: [email protected] (D. Pla).

Background: The timely parenteral administration ofantivenom is the most effective treatment for the





neglected tropical pathology of snakebite envenoming. Adeep knowledge of the toxin composition and theimmunological profile of venoms are central for devel-oping polyspecific antivenoms exhibiting broad para-specificity and cross-reactivity against the mostmedically-relevant snakes of a given geographical area.Preclinical neutralization tests and antivenomic assess-ments are necessary to demonstrate antivenom safety andefficacy prior to clinical trials. Antivenomics is a proteomictool for the qualitative and quantitative analysis of theimmunoreactivity of antivenoms. The original (firstgeneration, 1G) antivenomics protocol is based on theimmunodepletion of toxins upon incubation of wholevenom with purified antivenom IgGs, followed by theaddition of a secondary antibody or immobilized IgG-binding moiety. Antivenom immunoreactivity is inferredindirectly through the proteomic characterization of thetoxin fraction that remains in solution after immunopre-cipitation. This antivenomic approach is not appropriatefor F(ab’)2 antivenoms.

Objective: (i) To design a newmethod based on affinitychromatography which allows assessing F(ab’)2 anti-venoms, employing two F(ab’)2 monospecific (anti-Cerastes cerastes and anti-Macrovipera mauritanica) anti-venoms; (ii) to compare the 1G, immunoprecipitationprotocol, and the new, second generation (2G, affinitycapture) antivenomic approaches. These would assess theimmunological profile of the pan-African EchiTAb-Plus-ICP� whole IgG antivenom, whose immunoreactivitycharacteristics have been previously tested towards thevenoms of a panel of African viperid snakes and spittingcobras.

Methods: F(ab’)2 fragments or whole purified IgGmolecules were coupled to a NHS-activated Sepharose�.After incubating the matrix with the venom, the non-retained fraction and the immunospecific venom compo-nents were analyzed and quantified by reverse-phase HPLCfollowed by venomic analysis.

Results: The affinity capture protocol allowed analyzingboth types of antivenoms. Furthermore the pan-AfricanEchiTAb-Plus-ICP� antivenom showed qualitatively similarimmunoreactivity patterns using either antivenomicapproach. Although quantitative departures were noticedbetween both methods, these may be ascribed to differ-ences in calculating the relative amounts of the non-recognized venom proteins.

Discussion: Our results indicate that both 1G and 2Gantivenomic methods can be used interchangeably toinvestigate the in vitro immunoreactivity of antivenoms.An advantage of the affinity approach is the reusabilityof the affinity columns. Furthermore, the smootherbaseline in RP-HPLC chromatograms of affinity columnfractions allowed better resolution and a more accuratequantification of the antivenomic outcome than theoriginal 1G protocol. These features contribute to thegeneralization, economy and reproducibility of themethod.

Keywords: snake antivenom, antivenomics, immunodepletion, immu-noaffinity protocol.10.1016/j.toxicon.2012.04.232

232. Death Adder Envenoming Causes Neurotoxicity notReversed by Antivenom - Australian Snakebite Project(ASP-16)

Christopher I. Johnston 1, Margaret A. O'Leary 2,Simon G.A. Brown3, Bart J. Currie 4, Geoffrey K. Isbister 2

for the ASP investigators1 School of Medicine Sydney, the University of Notre Dame Australia,Darlinghurst, NSW, Australia2Department of Clinical Toxicology and Pharmacology, Calvary MaterNewcastle and the University of Newcastle, Newcastle, NSW, Australia3Centre for Clinical Research in Emergency Medicine, Western AustralianInstitute for Medical Research, Royal Perth Hospital and University ofWestern Australia, Australia4 Tropical Toxinology Unit, Menzies School of Health Research, CharlesDarwin University, Darwin, Australia

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

Background: Acanthophis spp (Death adders) occur inAustralia, Papua New Guinea, and parts of easternIndonesia and envenomingmainly cause neurotoxicity. Theobjectives of this study were to report the clinicalsyndrome of death adder envenoming and response toantivenom treatment.

Methods: Definite bites were recruited from theAustralian Snakebite Project (ASP) as defined by expertsnake identification or detection of death adder venom inblood by enzyme immunoassay. Clinical effects and labo-ratory results were extracted from the ASP database,including the time course of neurotoxicity and response totreatment. Enzyme immunoassay was used to measurevenom concentrations before and after administration ofantivenom.

Results: Twenty nine patients had definite deathadder bites with a median age of 45 years (Range: 7 to74y); 25 were male. The species of Death adder wasdetermined in nine cases: four A. praelongus (Northerndeath adder), two A. antarcticus (Common death adder),two A. hawkei (Barkly Tableland death adder) and one A.rugosus (Rough-scaled death adder). Envenomingoccurred in 14 patients. Two further patients had allergicreactions without envenoming; both were snakehandlers with previous death adder bites. Of 14 enve-nomed patients, 12 developed neurotoxicity charac-terised by ptosis (12), diplopia (9), bulbar weakness (7),intercostal muscle weakness (3), limb weakness (6). Tworequired intubation. Only 2 of the 12 had non-specificsystemic symptoms. One patient bitten by a Northerndeath adder developed myotoxicity and one patient onlydeveloped systemic symptoms (abdominal pain andvomiting) without neurotoxicity. No patients developedcoagulopathy. The median peak venom concentration in17 patients with pre-antivenom bloods available was9.3ng/mL (interquartile range 4.4-24ng/mL, range 0.7-245ng/mL). Antivenom was administered to 14 patientswho all received an initial dose of one vial of death adderantivenom. Subsequent doses were administered in eightpatients. In eight patients where post-antivenom bloodsamples were available, no venom was detectable afterone vial of antivenom. In all 12 patients treated forneurotoxicity, persistent neurotoxicity occurred for 5 to168 hours after antivenom.



Conclusion: Death adder envenoming is characterisedby neurotoxicity. One vial of death adder antivenom wassufficient to bind all circulating venom. The persistence ofneurotoxic effects after antivenom suggests that neuro-toxicity may not be reversed by antivenom.

Keywords: Death adder, envenoming, antivenom, neurotoxicity10.1016/j.toxicon.2012.04.233

233. Hospital Based Retrospective Study of SnakebiteEpidemiology in Western Development Region of Nepal

Chhabi L Thapa 1, Kamal Devkota 2, Dev P. Pandey 3

1District Health Office, Sindhulimadi, Sindhuli, Ministry of Health, NepalGovernment, Nepal2Central Department of Zoology, Tribhuvan University, Kirtipur, Nepal3Biodiversity and Climate Research Center, Frankfurt, GermanyE-mail address: [email protected] (D.P. Pandey).

Background: Snakebite is a common and life threat-ening public health problem in Nepal. Epidemiological dataare fragmentary and sparse in Nepal and accurate,comprehensive epidemiological data on snakebites is stilllacking. This study sought to characterize the snakebiteepidemiology in Western Development Region of Nepal.

Methods: A retrospective study of three years’ (2008-2010) snakebite data from medical records of 10 healthinstitutes (1 Zonal Hospital, 3 District Hospital, 1 PrivateHospital, 1 Mission Hospital, 2 Primary Healthcare Centersand 2 Army Camps) in the Western Development Region ofNepal was carried out during June 2011 to February 2012 bythe use of pretested data sheets. Snakebite data weremanually searched from those health institutions wheresnakebite victims used to be reported and governmentsupplied anti-snake venom freely. Snakebite reported inmedical records based on an eye witness and/or claim ofsnakebite victim, brought snakes, clear signs of snakebitewounds and symptoms of snakebite envenomation wereincluded in the study. Those with a claim of snakebite withno or poor proof of snakebite were considered as suspectedsnakebite and not included in analysis. Statistical analysiswas done by the application of MS Excel and R. The studywas approved by the Ethical Clarence Review Board inNepal Health Research Council.

Results: The overall confirmed snakebite reported inthree years was 6,993 of which 640 (9%) were envenomedand treated with antivenom (an average of 16 ASVS vialswere administered to each victim) and the suspectedsnakebite cases were 2,562. The overall case fatality ratewas 13% (10% in 2008, 16% in 2009, and 13% in 2010). Julyand August was the highly snakebite risk months (21% ofoverall snakebites in each month) in this region. Themajority of snakebites were reported between 15:00 and21:00 hours.

Discussion: Snakebite and envenomation were greaterin 2010 than in the two previous years. But the case fatalityrate was greatest in 2009. This study detected decreasedmortality associated with anti-snake antivenom use in thisregion.

Conclusion: Snakebite records in the existing snakebitetreatment center were poor and prevented complete

characterization of the epidemiology of snakebite epide-miology in the Western Development Region of Nepal.Antivenom use was associated with decreased mortality.

Keywords: Envenomation, case fatality, anti-snake venom10.1016/j.toxicon.2012.04.234

234. Severity of Snakebites in Children in the UnitedStates: 2000-2009

Scott A. Letbetter 1, Sharla A. Letbetter 1,2, David L. Morgan 2

1 Scott and White Memorial Hospital, Dept of Emergency Medicine, Temple,TX, USA2 Texas A&M Health Science Center, Temple, TX, USAE-mail address: [email protected] (S.A. Letbetter).

Background: Many of the 10,000 victims of snakebiteseach year in the US are under 18 years of age. The snakebitemanagement of this population has not been studied asextensively as adults; however, previous studies havedemonstrated that less than 2% of the pediatric victimsresult in major clinical effects. There may be particularcharacteristics specific to this population that could predicttheir overall outcomes. Treating physicians could use thisinformation to direct emergency management of thepediatric victims of snakebites.

Objective: Our goal was to determine the distinctcharacteristic differences of pediatric victims of snakebitesbetween those who had major and those who had minoroutcomes.

Methods: Observational, case-control study of tele-phone calls to all US poison centers (National Poison DataSystem) for human victims of snakebites under 18 yearsfrom 2000 to 2009. Major outcome was defined as“significant disability” or death. Minor outcome wasdefined as “minimally bothersome” or no clinical effect.Those with moderate outcome were excluded.

Results: There were 20,285 pediatric snakebites duringthe 10 year study period. Only 378 (1.9%) had a majoroutcome, 8,563 (42.2%) had a minor or no effect, and 11,344(55.9%) had a moderate or unknown effect and wereexcluded. There were 3 deaths from snakebite. Mostvictims of major (65.9%) and minor bites (72.3%) weremales (p ¼.008). Also, most victims of major (66.9%) andminor bites (78.9%) were over 5 years of age (p<.001).Seventeen States did not have a single snakebite thatresulted in a major outcome. Four states (Texas, Florida,Georgia and California) represent 53.2 % ofmajor outcomes.Most victims of major (78.8%) and minor bites (52.2%) werebitten by venomous snakes (P< .001). Nonvenomoussnakes caused 1.5% of major and 9.6% of minor outcomes(p<.001). “Unknown snakes” accounted for 20.1% of majorand 38.1% minor outcomes (p<.001). Rattlesnake bitesproduced 42.3% of major and 6.1% of minor outcomes(p<.001). Surprisingly, many major (44.1%) and minor bites(38.1%) occurred from September to April (p ¼.022).

Conclusions: This is the largest outcome analysis ofsnakebites in pediatric victims. Almost half of these victimshad only minor or no clinical effects. Significant disability ordeath of these victims was rare. Severity of outcome is asso-ciatedwith victim gender, age, geography, season, and type of




snake. These results may be useful for preventing majoroutcomes and the planning for snakebite management.

Keywords: Snakebite, pediatrics, outcomes, severity, USA10.1016/j.toxicon.2012.04.235

235. Antivenom Therapy Following Snakebite:Effectiveness and Strategies for Delivery in West Africa

Abdulrazaq G. HabibInfectious & Tropical Diseases Unit, Dept of Medicine, Bayero UniversityKano, Aminu Kano Teaching Hospital, Kano, NigeriaE-mail address: [email protected].

Background: In West Africa antivenoms have beenuseful in clinical management of snakebite envenoming.Response is often dramatic especially following carpetviper envenoming where restoration of blood coagulabilityand resolution of spontaneous haemorrhage is rapidlyachieved. In bites from certain snakes manifesting withsimilar coagulopathies elsewhere, antivenom effectivenessmay be less dramatic and its use has been questioned. Wetherefore critically evaluated clinical studies on envenom-ing and antivenom therapy conducted in the region todetermine their effects and explored the appropriatestrategies for delivery of care. Efficacy of antivenomsdeveloped from the region was explored from preclinicalstudies.

Methods: All observational, interventional andpreclinical studies conducted in the region (or on anti-venoms derived from the region) were systematicallyevaluated to determine the effect of antivenoms used in thestudies. Effectiveness of antivenoms in resolving features ofenvenoming or curtailing mortality was determined wherefeasible. Different strategies for optimally delivering anti-venom therapy in rural savanna populations was exploredand compared. Published data was re-analyzed.

Results: There were 23 studies reported with evaluableinformation on aspects of antivenom therapy from theregion. There were 12 observational studies (8 non-comparative and 4 comparative clinical studies), 4comparative clinical trials and 7 preclinical studies. 13 ofthe 16 clinical studies determined and strongly suggestedeffectiveness of antivenom therapy. From 3 of thecomparative studies: regionally appropriate antivenomtherapy conferred protection against deaths by 87% (95%CI:52-97%) when compared to inappropriate therapy, by 83%(95%CI: 4-97%) when compared to no therapy, and by 90%(95% CI: 55-98%) when combined with staff education andstandardization of clinical management. In preclinicalstudies most antivenoms used in practice have activityagainst venoms of local snakes and effect may be broaderextending to more species in larger areas of Sub-SaharanAfrica. Modality of antivenom delivery usually followsa ‘centralized’ district-peri-urban approach while ‘de-centralized’ hub-and-spoke rural model which reducesdelay (found to predispose tomortality) found to access hasnot been used commonly.

Conclusion: Antivenom therapy is effective in manage-ment of snakebite especially carpet viper envenoming in

West Africa. While a placebo controlled trial will be uneth-ical, antivenoms confer protection of over 80% against deathin observational studies. Strategies for improving careshould include utilizing regionally appropriate antivenoms,educating staff and standardizing clinical care. Centralizedversus de-centralized modes of antivenom delivery shouldbe explored further to determine respective benefits, risks,costs and feasibility of the approaches.

Keywords: Antivenom, effectiveness, West-Africa10.1016/j.toxicon.2012.04.236

236. Determinants of High Cost of Care Among Victimsof Snake Bite in Kaltungo, Gombe State, Nigeria, 2009

Mahmood M. Dalhat 1, Hamza Muhammad 1,Saidu B. Abubakar 2, Iliasu Garba 1, Ibrahim M. Yola 1,Abdulrazaq G. Habib 1

1 Infectious and Tropical Diseases Unit, Department of Medicine, Aminu KanoTeaching Hospital, Kano, Nigeria2Kaltungo General Hospital, Kaltungo, Gombe State, NigeriaE-mail address: [email protected] (H. Muhammad).

Background: Envenomation by Echis ocellatus (carpetviper) is an important cause of morbidity and mortality inWest Africa. Inaccessibility to care for snake bites results inineffective and harmful remedies. We conducted a crosssectional study of cases that presented in October 2009 toKaltungo General Hospital. Our objective was to identifythe determinants of high cost of care among victims ofsnake bite.

Methods: We administered questionnaires to all sus-pected cases that attended Kaltungo General Hospital inthe October 2009. We defined a suspected case as anypatient who presented to the hospital with history of snakebite during the study period. We obtained further infor-mation from clinical examination and hospital recordreview. We collected information on patient demographicfactors, type of snake, time from bite to presentation,clinical signs and symptoms, first aid given before presen-tation, duration of hospital stay, estimated cost of care,average family monthly income, and final outcome.

Results: We documented 109 patients; 77% were male.Median age was 24 yrs (range 6-80yrs). The main source ofincome for the families was farming (78%). Delayedpresentation to the snake bite centre of >24hrs wasrecorded by 26 cases (24%). On univariate analysis, patientswith delayed presentation (>24hrs) where more likely toincur high cost of care (OR: 7.4; 95% CI: 1.6 – 20.8), bleed(OR: 3.6; 95% CI: 1.4 – 9.5), ingest herbs (OR:12.7; 95% CI:1.6 – 98.9), apply traditional ailments at site of bite (OR:12.1; 95% CI: 1.6 – 93.7), or incise bite (OR: 2.8; 95% CI: 1.1 –

7.5). Risk factors for high cost of care where: delayedpresentation, hospital stay >2days (OR: 14.6; 95% CI: 1.9 –

115) and bleeding (OR: 4.0; 95% CI: 1.5 – 20.5). No statis-tically significant relationship between delayed presenta-tion and sex (OR: 1.7; 95% CI: 0.6 – 4.6), pediatric age group(OR: 1.6; 95% CI: 0.6 – 4.6) or being a farmer (OR: 0.8; 95%CI: 0.3 – 2.1). Multivariate analysis revealed delayedpresentation (>24hrs) (aOR: 5.8; 95% CI: 2.0 – 17.0) and




hospital stay >2days (aOR: 19.5; 95% CI: 2.0 – 192.3) asindependent risk factors of high cost of care.

Conclusion: Most cases of snake bite occur in youngproductive farmers and could impact local food production.Delay in presentation associated with unorthodox, harmfulpractices, could result in prolonged hospital stay and highcost of care.

Fig. 1. A. Haematoma from bite on the buttocks; B. Leg swelling; C. Cellulitisand gangrene; D. Bleeding diathesis.

Keywords: Access, cost of care, Echis ocellatus, envenomation, Nigeria10.1016/j.toxicon.2012.04.237

237. Snakebite Survivors Club: Ten-year, retrospectivereview of Crotaline envenomations in Central California

Susanne Spano 1, Fernando Macias 1, Brandy Snowden 1,Rais Vohra 1,2

1UCSF-Fresno Medical Center, Fresno, CA, USA2California Poison Control System, Fresno-Madera Division, Madera, CA USAE-mail address: [email protected] (R. Vohra).

Objective:We investigated clinical patterns of Crotalineenvenomation presenting to a tertiary-care academichospital in Central California over a 10-year period.

Methods: An IRB-approved, retrospective chart reviewwas conducted on all patients diagnosed with snakebitefrom December 2000 to December 2010. Data abstracted:demographics, anatomic location of bite, comorbid condi-tions and intoxicants, length of stay, antivenom dose,laboratory results, and complications or procedures.

Results: There were 46 snakebite cases admitted overthe study period. Five were “dry bites;” the remainingcases (41/46) received antivenom. There was a male

predominance (83% male victims). Upper extremity biteswere more common (32/41 upper vs 10/42 lowerextremity). One victim sustained bilateral bites to thehands. Thirty-five patients (85%) were admitted, with anaverage length of stay 2.12 days. The longest hospitalizationwas 15 days. There were no fatalities. The average timefrom bite to ED presentation was 164 minutes. Bitesoccurred during every month except November, with themajority occurring during spring and summer months andpeaking in June (12/42 cases). Most bites occurred in thehours between noon and 8 pm. The amount of antivenomgiven ranged from 2 to 35 vials (average, 9 vials). Interfa-cility transfers were common in our study population:thirteen (32%) patients were transferred into our emer-gency department for a higher level of care, and 3 (7%) weretransferred out (two because of insurance requirements,and one for higher level of Pediatric ICU care). There wereno surgical interventions in our study group. Intoxicationdid not appear to play a major role in this population asonly 3 patients (7%) were found to be acutely intoxicated:one with cannabis and amphetamines, 1 with alcohol, and1 with opioids.

Conclusions: In Central California, Crotaline enveno-mations occurred mainly in adult males. Dry bites, or bitesnot requiring antivenom administration, were uncommon,comprising only 10% of bites in this study population.Contrary to popular and clinical beliefs, substance abuseand/or alcohol intoxication did not appear to play a role inthe majority of patients. Care providers and snakebitespecialists should also be aware that snakebite patients areoften transferred between facilities, a finding that may beuseful in informing future first aid protocols and research.We hope these findings add concrete data and help correctsome common misconceptions about snakebites in CentralCalifornia.

Keywords: Snakebites, epidemiology, interhospital transfers10.1016/j.toxicon.2012.04.238

238. Incidence and Management of Snakebite inNorthern Central African Republic

Séverine Gras 1, Gaëtan Plantefève 2,Jean-Philippe Chippaux 3

1Département d'Anesthésie - Réanimation, Fondation OphtalmologiqueAdolphe de Rothschild, Paris, France2Réanimation Polyvalente, CH Victor Dupouy, Argenteuil, France3UMR 216 “Mère et enfant face aux infections tropicales”, Institut deRecherche pour le Développement and Université Paris Descartes, SorbonneParis Cité, Faculté de Pharmacie, Cotonou, BéninE-mail address: [email protected] (J.-P. Chippaux).

Background: Snakebite represents a serious publichealth problem in sub-Saharan Africa.

Methods: A retrospective studywas conducted in Paouahospital (northern CAR) for 27 months to assess the inci-dence and severity of snakebites. A team of Médecins SansFrontières (MSF) is working in this hospital the resources ofwhich are better than the average of sub-Saharan facilities.

Results: 842 people were registered for snakebite ofwhich 825 were included in the study. The seasonal




distribution shows a slight increase during rainy season.Only 11 snakes were identified. The sex ratio was 1.56 (503M/322 F) and the median age [IQ:25;75] was 17 [10;27.5]years. A third of the patients (174/523) reached the hospitalwithin 6 hours after the bite, while 46.5% (243/523) arrived12 hours after or more. Feet were involved in 70.7% of casesand hands in 24.6%. Immunotherapy was administered to644 patients (78.1%) who received 1.9 �0.06 [IC:95%] vials.Antivenom was renewed twice or more for 131 patients(20.3), owing to edema progression (40%), persistentbleeding (30%), abnormal coagulation test (30%) or persis-tence of neurological disorders (0.2%). Three patients didnot benefit from immunotherapy due to lack of antivenom.The average staywas 4.03�0.28 days. Seven patients (0.8%)showed clinical signs consistent with envenoming byelapid. Viper-like envenomation was observed in 640patients (77.6%). Edema was present in 681 of 812 patientsfor whom the data was specified (83.9%). Edema involvingtwo joints was observed in 399 patients (49.1%) and edemareaching or exceeding the limb's root was present in 114(14%). A clinical sign of hemorrhage (blisters, persistentlocal bleeding, epistaxis, gingival bleeding, hemoptysis,hematemesis) was reported in 145 patients (17.6%). Fivedeaths (0.6%) occurred respectively in 4 children and 1adult. One of the children died without obvious signs ofenvenomation probably resulting from poisoning by plantsused by traditional healer. Two other children arrived at thehospital with a severe anemia and died despite transfusionand antivenom. The fourth died from a sepsis. The adultdied from a severe hemorrhagic syndrome withoutreceiving antivenom due to out of stock. Local complica-tions (necrosis, abscess, flessum) occurred in 68 patients(10.6%). Surgeries were required in 29 for debridement(23 patients), skin graft (4 patients) and amputation(2 patients).

Conclusion: Better management of snakebites andbroad use of immunotherapy could explain the goodresults.

Keywords: Snakebite, antivenom, Africa10.1016/j.toxicon.2012.04.239

239. Combined Neurotoxicity and Hematotoxicity withClinically Significant Bleeding after Mohave Rattlesnake(Crotalus scutulatus) Envenoming in SouthernCalifornia

Sean P. Bush, Eric T. Teacher, Linda Daniel-Underwood,Sarah R. Pearl, Joshua Westeren, Tammy H. Phan,Ellen ReiblingLoma Linda University School of Medicine, Department of EmergencyMedicine, Loma Linda, CA, USAE-mail address: [email protected] (S.P. Bush).

Methods: Case report. The snake's head was examinedby 4 herpetologists with unanimous agreement onspeciation.

Results: We describe a confirmed Mohave rattlesnakeenvenomation with angioedema, hemorrhage, neurotox-icity and rhabdomyolysis. A 31-year-old male presented 1

hour after being bitten by a Mohave rattlesnake encoun-tered in Adelanto, California. On arrival he was tachycardic,hypotensive and altered. He had minimal tissue effects atthe bite site. However, he had facial and airway angioe-dema. He began to complain of progressive difficultybreathing, swallowing and had extraocular muscle weak-ness consistent with neurotoxicity. He also developedgeneralized myokymia. He then had two episodes ofhematemesis. The patient was intubated and airway wasnoted to be edematous during the procedure. Because thepatient was in shock with serious active bleeding, an initialdose of 12 vials of CroFab � was initiated. He continued toshow signs of hemorrhaging with multiple large bloodybowel movements (approximately 1.5 L), a 8-gram drop inhemoglobin, bloody drainage from foley / gastric tubes andconjunctival hematomas. Initial labs confirmed coagula-pathy, INR 3.2, Fibrinogen <50 mg/dl. First platelet countwas normal, but severe thrombocytopenia developedshortly thereafter. He was transfused and admitted to theICU. He ultimately received 6 units of PRBCs, 5 units of FFPand 30 vials of CroFab�, which resolved his bleeding andeventually his coagulopathy as well. He developed rhab-domyolysis, which responded to fluid therapy. He wasextubated on day 4, and discharged on day 9 with normallabs andwas fully intact neurologically by day 11 follow-up.

Discussion: Until now, only Mohave rattlesnakes withVenom A (neurotoxic) venom have been reported inSouthern California. Venom B (hemorrhagic and tissue-destructive) effects have not been previously describedafter bites by this species from this region. Other areas,such as Arizona and Texas, may have snakes with A, B orAþB venom/effects. In California, this is a novel find.

Conclusion: Hematotoxicity with clinically significantbleeding, together with neurotoxicity and other seriousvenom effects, can be seen after Mohave rattlesnakeenvenoming in Southern California.

Keywords: Mohave, Mojave, venom10.1016/j.toxicon.2012.04.240

240. Development of a Double Sandwich FlourescentELISA to Detect Rattlesnake Venom In BiologicalSamples from Horses with a Clinical Diagnosis ofRattlesnake Bite

Lyndi L. Gilliam 1, Amy Giulioli Canida 2,Dianne McFarlane 3, Todd C. Holbrook 1, Mark Payton 4,Charlotte L. Ownby 5

1Oklahoma State University Center for Veterinary Health Sciences,Department of Veterinary Clinical Sciences, Stillwater, Oklahoma, USA2Banfield Animal Hospital, Oklahoma City, Oklahoma, USA3Oklahoma State University Center for Veterinary Health Sciences,Department of Physiological Sciences, Stillwater, Oklahoma, USA4Oklahoma State University, Department of Statistics, Stillwater, Oklahoma,USA5Oklahoma State University, Office of the Vice President for Research andTechnology Transfer, Stillwater, Oklahoma, USAE-mail address: [email protected] (L.L. Gilliam).

Background: Themechanism of cardiotoxicity in horsesfollowing envenomation by rattlesnakes endemic to NorthAmerica is unknown.




Objective: We hypothesized that larger venom doseswould cause more severe cardiac damage. The objective ofthis study was to develop an ELISA to detect rattlesnakevenom in equine biological samples in order to test thishypothesis.

Methods: Nineteen horses with a clinical diagnosis ofrattlesnake bite were enrolled. Cardiotoxicity wasassessed using serum troponin and the presence ofarrhythmias on ECG. A double sandwich fluorescentELISA was developed to detect rattlesnake venom inurine and bite site swabs collected from these horses. Abite site swab was taken at admission and urine wascollected at presentation, 24, 48, 72, 96 hours, 1 week and1 month post presentation. Urine was available from 19horses however not at every time point. Bite site sampleswere available from 9 horses. Samples were consideredpositive if venom concentration was >2 standard devia-tions above the negative control.

Results: Venom was detected in urine of 13 of 19horses and in bite site samples from 5 of 9 horses. Nocorrelation was found between venom concentration andcardiotoxicty.

Discussion: Venom elimination in urinewas found to behighly variable. With limited sample sets it was notpossible to determine peak elimination or maximumvenom concentration.

Conclusions: An ELISA was developed that successfullydetected rattlesnake venom in equine biological samples.More consistently timed urine collection may be necessaryto further investigate the relationship between venom doseand cardiotoxicity.

Keywords: Rattlesnake, horse, ELISA10.1016/j.toxicon.2012.04.241

Fig. 1. Fang, in vivo

Fig. 2. Fang length

241. Juvenile Rattlesnake Fang as a Retained ForeignBody: Clinical Diagnosis

Joshua J. Ennis 1, Farshad Shirazi 1,21University of Arizona, Dept. of Emergency Medicine, Tucson, AZ, USA2Arizona Poison and Drug Information Center, Tucson, AZ, USAE-mail address: [email protected] (J.J. Ennis).

Background: An estimated 6,000-8,000 snakebitesoccur each year in the U.S. with more than half comingfrom venomous species, prompting many calls to poisoncenters. The Arizona Poison and Drug Information Center(AZPDIC) lies in a unique location in the southwesterndesert region of the United States, handling approximately200 calls around the state related to rattlesnake even-omation. Recently a surgeon asked AZPDIC regarding theability to see a juvenile rattlesnake fang in tissue, as he hada patient bitten by a juvenile rattlesnake whose examsuggested a retained fang. To our knowledge it wasunknown if this could be diagnosed by traditional diag-nostic modalities.

Objective: To determine if a retained juvenile rattle-snake fang is identifiable as a foreign body with traditionalclinical imaging techniques.

Methods: Extraction of a fang from a post-mortemjuvenile rattlesnake Crotalus Atrox, popularly knownas the Western Diamondback rattlesnake, aged atless than 1 year of age, followed by implantation indiffering media with ultrasound and radiography thenperformed.

Results: Juvenile rattlesnake fang was visualized withboth ultrasound and radiography.

Conclusions: Juvenile rattlesnake fang as a retainedforeign body is clinically diagnosable with ultrasound andradiography, and may assist in securing this diagnosis ina clinical setting although small fang size, ultrasoundmachine resolution, and acoustic windows are limitingfactors.


Fig. 3. Radiograph of fang, in pig.


Fig. 4. Fang, long axis.

Keywords: Snake, envenomation, fang10.1016/j.toxicon.2012.04.242

242. The Effect of Pre-hospital Care for VenomousSnakebite on Outcome in Nigeria

Michael C. Godpower 1, Thacher D. Thomas 2, Shehu Mil 11Department of Family Medicine, Aminu Kano Teaching Hospital, Kano,Nigeria2Department of Family Medicine, University of Jos, NigeriaE-mail address: [email protected] (M.C. Godpower).

Background: Snakebite is a common but neglectedhealth problem in rural sub-Saharan Africa. In some ruralNigerian hospitals up to 50% of the total bed capacity may

be occupied by snakebite victims at peak times of the earlyrainy season and harvesting periods. The untreatedmortality (without antivenom) is 10-20%. Echis ocellatus(carpet viper) is responsible for 66% of bites in the Nigeriansavanna. Various pre-hospital care of doubtful efficacy arereceived by victims.

Methods: We prospectively studied 72 consecutivesnakebite victims at a rural north central Nigerian hospital.The primary outcome assessed was death or disability athospital discharge.

Results: Victims were predominantly male farmers,and in 54 cases (75%) the snake was identified as carpetviper (Echis ocellatus). Most subjects (58, 81%) attemptedat least one first aid measure after the bite, includingtourniquet application (53, 74%), application (15, 21%) oringestion (10, 14%) of traditional concoctions, bite siteincision (8, 11%), black stone application (4, 5.6%), andsuction (3, 4.2%). The majority (44, 61%) presented late(after 4 hours). Most (53, 74%) had full recovery at hospitaldischarge. Three deaths (4.2%) and thirteen (18%)disabilities (mainly tissue necrosis) occurred. The use ofany first aid was associated with a longer hospital staythan no use (4.6�2.0 days versus 3.6�2.7 days, respec-tively, P¼0.02). The antivenom requirement was greater insubjects who used a tourniquet (P ¼ 0.03) or presentedlate (P ¼ 0.02). Topical application (Odds Ratio 15, 95% CI1.4-708) or ingestion of traditional concoctions (OR 20,95% CI 1.4-963) was associated with increased risk ofdeath or disability. Ingestion and application of concoc-tions were associated with a longer time interval beforepresentation, a higher cost of hospitalization, and anincreased risk of wound infection.

Conclusion: Traditional first aid measures for viperbites, like use of tourniquets and traditional concoctions,potentially contribute to morbidity and mortality in ruralNigerian communities. Delays in antivenom administrationdue to late presentation and erratic antivenom supply arealso additive factors. Community education on appropriateactions after snake bite, including limb immobilization,cleansing the bite site with soap and water and rapidtransport of victims to the hospital could reduce thecomplications from viper bites in Africa.

Keywords: Viper, envenomation, first aid10.1016/j.toxicon.2012.04.243

243. Continuous Crotalidae Polyvalent Fab (Ovine)(FabAV) for Selected Snakebite Patients

Sean P. Bush 1, Steven A. Seifert 2, Susan D. Smith 1,Tammy H. Phan 1, Sarah R. Pearl 1, Ellen E. Reibling 1

1Department of Emergency Medicine, Loma Linda University School ofMedicine, Loma Linda, California, USA2Department of Emergency Medicine, University of New Mexico School ofMedicine, and New Mexico Poison and Drug Information Center,Albuquerque, New Mexico, USAE-mail address: [email protected] (S.A. Seifert).

Background: In patients bitten by rattlesnakesand treated with Crotalidae Polyvalent Immune Fab(Ovine) (FabAV), persistent or recurrent hematologic effects




(hypofibrinogenemia, prolonged PT/INR, prolonged PTT,and/or thrombocytopenia) are common, difficult tomanage, and may result in morbidity and mortality. Theoptimal management of persistent or recurrent hemato-logic abnormalities, particularly the use of further treat-ment with antivenom, has not been well defined.

Objectives: To describe experience using a continuousinfusion of FabAV for persistent and/or recurrent hemato-logic effects in rattlesnake envenomation.

Methods: Retrospective, observational case series.Repeat bolus doses as well as continuous infusions of FabAVwere used on an inpatient basis for patient benefit to try toreverse persistent or recurrent hematologic abnormalitiesand/or associated bleeding complications. Indications,dilution and infusion protocols, and duration of therapywere individualized.

Results: Five cases were identified between July 2010and September 2011. All patients had profound hemato-logic abnormalities that persisted, recurred and/or wereassociated with serious bleeding. Several patients receivedrepeat bolus infusions of FabAV, with or without bloodproducts, with either inadequate or only transient benefi-cial response. All patients were then managed witha continuous infusion of FabAV and all responded to thecontinuous infusion of FabAV, titrated to effect, withcessation of progression and, in most cases, improvementin hematologic abnormalities. Rates of infusion varied from2 to 4 vials per 24 hours (mean ¼ 3.3 þ/- 1.0 vials/day) forcontrol or reversal of hematologic abnormalities. Theduration of FabAV infusion was between 4 and 8 days fromthe time of envenomation (mean ¼ 6 þ/- 2 days), afterwhich hematologic values were normalized or werenormalizing and continued to do so.

Discussion: The use of FabAV as a continuous infusion,particularly after the acute phase of envenomationhas passed, provides a continuous source of circulatingantibodies to neutralize venom components reachingcirculation from tissue stores and allows natural replen-ishment of hematologic factors such as platelets and/orfibrinogen. This method is the most efficient use ofFabAV, avoiding the wasteful excess of a bolus dose, maybe more effective, eliminating the potential for destruc-tion of hematologic factors when protectiveantivenom levels are lost between bolus FabAV doses,and appears to be safe. Further assessment of thestability and sterility of the product is needed andsequential IVs over 4 to 6 hours are currently recom-mended rather than a single infusion over 24 hours. Theneed to continue hospitalization for its administration isthe major drawback, but that may be needed for otherindications (e.g. bleeding) and this method is best suitedfor event.

Conclusions: A continuous infusion of FabAV between2 and 4 vials per day, titrated to effect, and continued for4 to 8 days post-envenomation was associated withreversal of persistent and/or recurrent hematologiceffects of rattlesnake envenomation and, combined withblood products, control of significant bleeding. Contin-uous infusion of Fab antivenom may be safer, more effi-cacious, and more cost-effective than observation

without FabAV treatment or as-needed dosing in high-risk patients.

Keywords: FabAV, continuous IV infusion, recurrence, hematologic effects,Crotalinae, envenomation10.1016/j.toxicon.2012.04.244

244. Mulga snake (Pseudechis australis) Bites; A Reviewof Significant Cases Including an Exceptionally SevereLocal Envenoming

Julian White 1, Scott Weinstein 1, Sam Alfred 1,2

1 Toxinology Dept., Women's & Children's Hospital, North Adelaide SA,Australia2 Emergency Department, Royal Adelaide Hospital, Adelaide SA, AustraliaE-mail address: [email protected] (J. White).

Background: The mulga snake (Pseudechis australis) isAustralia's largest venomous snake: adults commonlyexceed 2m in length. The species is common throughoutmuch of inland and northern Australia. Unlike most otherdangerous Australian snakes, mulga snakes cause antico-agulant coagulopathy, but the most prominent feature ofenvenoming is severe systemic myolysis.

Method: Cases of mulga snake bite treated by one ormore of the authors over the last 30 years were reviewed,with regard to circumstances of bites, local and systemiceffects, outcomes of treatment, based on prospectivelycollected data at the time of each bite.

Results: Mulga snake bites occurred in the warmermonths of the year, with both diurnal and nocturnalencounters, including bites inflicted while the victim wasasleep in bed. Envenoming was systemic in virtually allcases, with predominantly myolysis manifest as massiveelevations of plasma CK, usually with macroscopic myo-globinuria. Anticoagulant coagulopathy was less commonand was not associated with clinical bleeding. Local painand swelling of the bite site and adjacent limb was a hall-mark feature. Symptomatic response occurred after 1 vialof suitable antivenom in most cases.

Case Report: An 18 yr old woman was bitten on theupper thigh by a large snake while in bed in a rural settinghouse. The snake was later identified as a mulga snake,confirmed by venom detection. A tourniquet was appliedas first aid. Retrieval was organised back to a majorhospital, and on arrival she had severe local pain and anarea of damaged skin around the bite site, with moderatesystemic myolysis and mild anticoagulant coagulopathy.She was given IV Black Snake antivenom (CSL Ltd., Mel-bourne) and although the systemic features resolved, thelocal wound progressed to full thickness skin lossrequiring debridement and secondary repair. This consti-tutes an unusually severe local reaction to mulga snakebite.

Discussion: Mulga snake bites are associated withwarmer weather, may occur at night and inside dwellings,as well as diurnally, with hallmark features of significantlocal pain and swelling, severe systemic myolysis, occa-sional anticoagulant coagulopathy, usually without clinicalbleeding, and respond well to a single vial of appropriate



antivenom. They can cause severe local tissue injury, but todate, this is an uncommon phenomenon.

Keywords: Mulga snake, snakebite, envenoming10.1016/j.toxicon.2012.04.245

245. Pressure Bandaging with Immobilization inCrotalinae Envenomation Controversy

Steven A. Seifert 1,2, Julian White 3, Bart J. Currie 4,Eric J. Lavonas 5

1New Mexico Poison and Drug Information Center, Albuquerque, NM, USA2 School of Medicine, University of New Mexico, Albuquerque, NM, USA3 Toxinology Department, Women's and Children's Hospital, North Adelaide,SA, Australia4 Tropical Toxinology Program, Menzies School of Health Research, CharlesDarwin University, Darwin, Northern Territory, Australia5Rocky Mountain Poison and Drug Center, Denver, CO, USAE-mail address: [email protected] (S.A. Seifert).

Background: In 2010, the American Heart Association(AHA) and American Red Cross (ARC) published theirupdated Guidelines for First Aid. Those guidelines, inten-ded to be applied by bystanders or by the victim, includedthe statement that pressure bandaging with immobiliza-tion (PBI) had been demonstrated to be effective for bitesby non-neurotoxic (Crotalinae) American snakes. This rep-resented a radical change in recommendations inmanaging Crotalinae envenomations pre-hospital in theU.S. In response to this, a number of individuals withexpertise in treating Crotalinae snakebite wrote to the AHA/ARC, questioning their analysis of existing data, theirconclusion that this method had been demonstrated to beeffective, and expressing concerns that, based on studies ofthis method elsewhere in the world, plus limited experi-mental animal data demonstrating elevated tissue pres-sures in Crotalinae envenomations, it was also potentiallyharmful. The AHA/ARC defended their process andconclusions, and refused to publish any of the letters or torevise their recommendations.

Methods: A position statement regarding PBI in Crota-linae envenomation was drafted and circulated to theAmerican College of Medical Toxicology, the AmericanAcademy of Clinical Toxicology, the American Associationof Poison Control Centers, the European Association ofPoison Control Centres and Clinical Toxicologists, theInternational Society on Toxinology and the Asia PacificAssociation of Medical Toxicology.

Results: The position statement reviewed the currentstate of the science on PBI and concluded, “The use ofpressure immobilization for the pre-hospital treatment ofNorth American Crotalinae envenomation is not recom-mended”. The position statement was endorsed by all sixorganizations. In addition, a Commentary was written toaccompany the position statement. The Commentary notedthe extraordinary concurrence of mainstream medicalopinion among experts on four continents and concludedthat the proper grading of current evidence on PBI wasClass III: Evidence and/or general agreement that a proce-dure/treatment is not useful/effective, and in somecases may be harmful. Both the position statement and

accompanying Commentary were published simulta-neously in Clinical Toxicology and the Journal of MedicalToxicology. In response to the impending publication of theposition statement, the AHA/ARC engaged in further dia-logue, agreed that their guideline was not clear regardingthe snake groups, geographic locations and individualcircumstances in which PBI might be applicable, and thatthe data was insufficient to deem PBI safe and effective.They further agreed to revise the guideline, includingcontent experts from the position statement-sponsoringorganizations in future guideline development.

Conclusions: Introduction of new treatments forsnakebite, particularly first-aid recommendations, shouldbe evidence-based and include considerations of contextand real-world application. Collaborative action by tox-inologists and toxinology organizations helped to correcta flawed first-aid recommendation for Crotalinaeenvenomations.

Keywords: Pressure bandaging, PBI, Crotalinae, envenomation, compart-ment syndrome10.1016/j.toxicon.2012.04.246

246. Non-front-fanged Colubroids; A Current Analysis ofMedical Significance

Scott Weinstein, Julian WhiteToxinology Dept., Women's & Children's Hospital, North Adelaide, SA 5006,AustraliaE-mail address: [email protected] (J. White).

Background: Non-front fanged colubroids (NFFC)comprise about 70% of extant snake species and includeseveral taxa now known to cause lethal or life threateningenvenoming in humans. A growing number of other taxaare implicated in medically significant bites. Some of theseare increasingly entering the commercial snake tradeposing a currently unquantified risk.

Methods: The global literature was searched casereports of NFFC bites. These cases were assessed forevidence-based value, clinical detail and verified speciesidentification. These data were subjected to meta analysisand a hazard index was generated for select taxa.

Results: Case reports meeting the selection criteriawere identified for about 120 species. Of these a smallsubset, designated Hazard level 1, represented thosespecies well documented as having lethal potential andmanagement of these cases included specific therapy for 3species (antivenom; Dispholidus typus, Rhabdophis tiginis,R. subminiatus), whereas others in this group (Thelotornisspp.) are treated with replacement therapy (eg cry-oprecipitate/fresh frozen plasma/ packed red blood cellsetc) and supportive care. Evidence-based analysis posi-tively contraindicates the use of heparin, antifibrinolyticsand plasmapheresis/exchange transfusion. Hazard level 2/3species consisted of several taxa that have mixed qualitydata implicating these as causing rare systemic effects(eg Boiga irregularis, Philodryas olfersii, Malpolon mon-spessulanus). Recommended management may include useof acetylcholinesterase inhibitors (eg neostigmine) andwound care on a case by case basis. Hazard level 3 species




comprised a larger group capable of producing significantlocal effects only and this often is associated with a pro-tracted bite (eg Heterodon nasicus, Alsophis (¼ Borikenophis)portoricensis, Platyceps rhodoracis). Management isrestricted to wound care. Bites by Hazard level 4 speciesconsisted of the majority of surveyed taxa and theseshowed only minor effects of no clinical importance.

Discussion: This study has produced the mostcomprehensive evidence-based listing of NFFC snakestabulated against medical significance of bites, togetherwith best-practice management recommendations. Thiswill assist clinicians in managing bites by these snakes. Thiscritical analysis assumes increasing importance concomi-tant with the growing exposure to lesser known NFFCsnakes, particularly in captive collections. This growingexposure may uncover further species of significance in thefuture. Careful and accurate documentation of bites byverified species of NFFC snakes is required to increase theevidence base.

Keywords: Colubrid snakes, snakebite, envenoming10.1016/j.toxicon.2012.04.247

247. Envenomation by Daboia mauritanica Snakes inTiznit Province, Morocco: Report of Four Cases

Chafiq Fouad 1,6, Chrouqui Nadia 2, El Jaoudi Rachid 3,4,Fekhaoui Mohamed 5, Soulaymani Abdelmajid 6,Rhalem Naima 1,6, Mokhtari Abdelghani 6,Soulaymani-Bencheikh Rachida 1,4

1Morrocan Poison Control Center, Morocco2Hassan Premier Hospital – Tiznit, Morocco3Military Hospital of Instruction Mohammed V. Rabat, Morocco4Medicine and Pharmacy Faculty. Rabat, Morocco5Rabat Scientific Institute, Morocco6Genetic and Biometric Laboratory, Sciences Faculty, Ibn Tofail University,Kenitra, MoroccoE-mail address: [email protected] (C. Fouad).

Background: In Morocco, the incidence of snake bites isestimated at 0.2 for 100 000 inhabitants with an annualaverage of 5 deaths. The identification of the aggressorsnake remains problematic because of the lack of qualifiedtaxonomic expertise in treating practitioners.

Methods: This is the first report inMorocco of four casesof confirmed envenomation by Daboia mauritanica occur-ring in Tiznit province, in the south of Morocco. The iden-tification of the snake was achieved by the MoroccanPoison Control Center and confirmed by the Rabat ScientificInstitute.

Results: Two cases of snakebite were severe. Clinicalsymptoms were characterized by thrombocytopenia, lowblood pressure, compartment syndrome, and hemorrhagicsyndrome. For one of the two cases, local necrosis of thethumb, thenar and hypothenar areas was observed andboth of these cases received fasciotomy. The medicalmanagement included vasopressors, antibiotics, analgesics,transfusion and aponeurotomy. Envenomation wasmoderate (extensive swelling, non-life threateningsystemic symptoms) andminor (local swelling, no systemicsymptoms) in the third and fourth cases, respectively.

Outcomes in these cases were favorable after a shorthospitalization. There is currently no antivenom withcoverage of this species and no antivenomwas given in anyof these cases.

Discussion: Daboia mauritanica is a venomous viperspecies listed by the Rabat Scientific institute with a widenative distribution in Morocco. There have been noprevious published, documented case reports of enveno-mation by this snake. Venom-induced toxicity is charac-terized by its proteolytic, hemorrhagic and phospholipasicactivity. It also has fibrinolytic and myolytic (skeletal andcardiac muscle) activity. In order to minimize the numberof envenomations, the population in this region should beeducated on the risks of bites by Daboia mauritanica. Toimprove outcomes, Medical personnel in this region shouldbe aware of the spectrum of toxicities as well as themanagement techniques available to treat Daboia maur-itanica envenomations.

Conclusions: Increased awareness of the populationconcerning the existence of Daboia mauritanica in Tiznitprovince, the availability of skilled medical providers, andan available, effective serotherapy against this specieswould decrease the morbidity and mortality due to thisenvenomation.

Keywords: Envenomation, Daboia mauritanica, Tiznit province, SouthernMorocco10.1016/j.toxicon.2012.04.248

248. Venomous Mixtures, Gamma Irradiation andAntivipmyn Africa�

Guillermo de la Rosa 1, Carlos Olvera 1, Andrés Alagón 2,Epifanio Cruz 3, Alejandro Alagón 1

1 Instituto de Biotecnología, UNAM, Cuernavaca, Mor., Mexico2Rancho Ojo de Agua, Agua Fría, Pue., Mexico3 Instituto de Ciencias Nucleares, UNAM, México City, MexicoE-mail address: [email protected] (G. de la Rosa).

Background: Antivenoms are the only treatmentknown to be effective to treat snakebites. AntivipmynAfrica�, produced by Instituto Bioclon, is a polyvalentantivenom against Sub-Saharan snakes with demonstratedsafety and efficacy. It is manufactured starting with serafrom two groups of horses, one immunized with a mix ofviperid venoms (Bitis arietans, B. gabonica, Echis leucogaster,E. pyramidum and E. ocellatus) and the other to elapidvenoms (Naja haje, N. melanoleuca, N. nigricollis, N. pallida,Dendroaspis polylepis and D. viridis). Those complex venomimmunogens can be highly toxic to horses; especiallyduring early phases of immunization. Several papers reportthat high energy irradiation of venoms could be a practicalsolution for reducing toxicity of venoms while retainingtheir immunogenicity. Accordingly, we decided tosystematically investigate the effect of gamma radiation onboth venom mixes and in their ability to induce protectiveantibody responses in horses.

Methods: We irradiated, using a Cobalt-60 gamma-rayirradiator, both dry and frozen (dry ice) venom mixes andfound that they needed huge irradiation doses to altervenom proteins. However, with venoms in solution




(5 mg/mL, saline) and at room temperature, we foundgamma-irradiation doses that decreased the toxicity ofvenom mixes while the venom protein componentsremained soluble.

Results: Crude venommixtures had a LD50 (IV route, 18-20 g, CD-1 mice) of 1.15 and 0.48 mg/Kg for viperid andelapid mixes, respectively. The optimal irradiation dose–maximum dose that doesn't cause protein precipitation-for viperid venom mix was 3.1 kGy while that for elapidmix was 5.5 kGy. Under those conditions, the LD50 of theviperid venom mix decreased by a factor of 2.8, while thatfor the elapid mix diminished 3.7 times. Each irradiatedimmunization mix was used to immunize groups of threehorses. None of the horses developed systemic toxicity.Their pooled sera had neutralization potencies comparableto pooled sera collected from horses immunized withuntreated venom mixes.

Discussion: Gamma-irradiated detoxified venoms,under well controlled and studied conditions, are a safeand practical alternative for the production of horseserum with high neutralization potency against snakevenoms.

Funding: Partially supported by Instituto Bioclon, SA deCV and CONACYT (México).

Keywords: African snake venoms, gamma radiation, venoms toxoids10.1016/j.toxicon.2012.04.249

249. Experience with Crotalidae Polyvalent Immune Fab(Ovine) for a non-North American RattlesnakeEnvenomation

Sean P. Bush, Tammy H. PhanDepartment of Emergency Medicine, Loma Linda University School ofMedicine, Loma Linda, California, USAE-mail address: [email protected] (S.P. Bush).

Background: There is little experience using CroFab� tomanage non-North American pit viper envenomation.

Methods: We report a case using CroFab� for a SouthAmerican rattlesnake (Crotalus durissus vegrandis)envenomation.

Results: A 22-year-old male presented to a southernCalifornia hospital after a bite by an Uracoan rattlesnake tohis ring finger, which occurred at 01:30 on 08/13/2011. Thesnake was imported from Venezuela, and herpetologistsconfirmed species. Initial labs (CBC, PT/INR, fibrinogen, andCK) were normal except fibrinogen, which was 143 mg/dL(reference 200-400). He was treated with CroFab� 5 vialsand transferred to our emergency department. Uponpresentation to our facility 5 hours post bite, the patientcomplained of marked pain, swelling and tenderness to theantecubital fossa. He was nauseated and vomited once.Another 4 vials of Crofab� were given at 08:15. Afterwardslabs were significant for thrombocytopenia 52 bil/L (refer-ence 140-340) and fibrinogen 98 mg/dL . An additional 4vials of CroFab� were administered at 12:15 withimprovement of both platelet count- and fibrinogen level.At 20:55, the patient complained of increased pain andswelling to the bite area so an additional 4 vials were givenat 21:10. At 18:32, he complained of increased swelling and

pain to the left forearm and received another 4 vials ofCroFab� at 19:31. On day 3, all clinical and laboratoryparameters had stabilized and the patient was discharged.He followed up on day 5 and all labs were normal. Hereturned again on day 7 for a second follow-up, and wasfound to have thrombocytopenia recurrence, witha platelet count of 111 bil/L. Fibrinogen and hemoglobinwere normal. He was admitted to our observation unit and4 vials of CroFab� were administered. The next morning alllab parameters were improving and continued to do so.Patient was discharged home on day 9. He returned onceagain on day 11, at which time his labs were normal andlocal effects had completely resolved.

Discussion: CroFab� is FDA-approved for envenoma-tions by any North American pit viper even though it ismade using the venom from only four species. Antivenomsgenerally have varying degrees of cross-protectivity againstvenoms not utilized in their manufacture. Although testedin murine LD50 models, there is very little clinical experi-ence with CroFab� for envenomation by anything otherthan a US Crotalinae.

Conclusion: Further investigation of the use of CroFab�

in non-North American vipers is warranted.

Keywords: Crotalidae, exotic, Crotalus durissus vegrandis10.1016/j.toxicon.2012.04.250

250. Critical Shortage of Coral Snake Antivenom isImpacting Patient Care

Cynthia R. Lewis-Younger, Jeffrey N. Bernstein,Jay SchaubenFlorida Poison Information Center Network, USAE-mail address: [email protected] (C.R. Lewis-Younger).

Background: Envenomation by Micrurus fulvius is rare.Approximately half the bites reported in the US occur inFlorida. Since manufacture of North American Coral SnakeAntivenin� was discontinued in 2003, supplies have beendwindling. We report three cases of envenomation withdelayed treatment due to supply problems.

Case One: A 39 year old presented approximately 30minutes following a bite between his thumb and forefinger.He remained relatively asymptomatic for almost 14 hours,whereupon he developed dysphagia and ptosis. Antivenomwas believed to be unavailable. He was intubated andplaced on a ventilator 19 hours post-bite, at which time 6vials of antivenom were administered. Attempts to extu-bate him on days 3, 6 and 12 failed; requiring re-intubationeach time. He was successfully extubated on day 24 anddischarged home at 30 days post bite.

Case Two: A 6 year old was bitten on the right indexfinger 45 minutes prior to presentation. He had an episodeof emesis prior to arrival, but was asymptomatic at time oftransfer to a tertiary facility. Despite administration of 5vials of antivenom, he was intubated and placed ona ventilator due to difficulty handling oral secretions. Thehospital course was complicated by hypotension andaspiration pneumonia. Extubation and discharge occurredon days 13 and 15 respectively.




Case Three: A 41 year-old presented approximately fivehours after being bitten by a coral snake on the right 3rd

finger. Initial symptoms were interpreted as tongueswelling, potentially an allergic response. He was paralyzedand intubated, and placed on a ventilator; then transferredto a tertiary care facility. The patient was extubated afteradmission to the intensive care unit but almost immedi-ately needed reintubation due to respiratory failure. Hereceived 5 vials of antivenin. He remained intubated fora total of 9 days, was hospitalized for 19 days and dis-charged to a skilled rehabilitation facility.

Discussion: Rapid administration of antivenom isimportant for the prevention of respiratory paralysis. Allthree patients experienced delays in treatment due to realor perceived shortages of antivenom, necessitating costlyintensive care. Early administration of antivenom canprevent the clinical signs of envenomation, and patientsmay be discharged within 24 hours.

Conclusions: The shortage of antivenom has reacheda critical point. Patient care has been compromised due toinadequate supplies. The timely administration of anti-venom is life saving and prevents the need for costlyintensive care.

Keywords: Coral snake, antivenom, shortage10.1016/j.toxicon.2012.04.251

251. Acute Hypersensitivity Reaction FollowingAdministration of Crotalidae Polyvalent Immune FabAntivenom: A Case Report

Gus A. Gross, Olga A. Pudovka GrossGuadalupe Regional Medical Center, Dept. of Hospitalist Medicine, Seguin,TX, USAE-mail address: [email protected] (G.A. Gross).

Introduction: Crotalidae polyvalent immune Fab(ovine) (CroFab�) is commonly used in the treatment ofsymptomatic North American crotaline snake envenoma-tion. When approved by the U.S. Food and Drug Adminis-tration in 2000, the incidence of immediatehypersensitivity reaction rate from CroFab�was reported asup to 19%. Recent systematic literature review and meta-analysis showed that it appears to be lower than previouslyreported, at 0.08. We describe the case of acute hypersen-sitivity reaction to CroFab� in a patient bitten byCopperhead.

Case history: The present case study depicts the enve-noming of a 71-year-old female who was bitten on her leftindex finger. She presented in Emergency Room withcomplaints of severe extremity pain and puncture wound,surrounded with edema and ecchymosis. Approximately 2hours after being bitten, edema and ecchymosis had reachedpatient's shoulder. She complained of the metallic taste inher mouth as well. Patient's past medical history significantfor multiple medical problems. Patient had multiple drugallergies. No prior history of anaphylaxis/anaphylactoidreaction. No prior history of envenomation. Our treatmentteam decided it was in patient's best interest to proceedwith CroFab�. Patient was premedicated with

methylprednisolone and diphenhydramine. Infusion wasstarted with initial dose of 4 vials in 250 ml normal saline.Within seconds of infusion of CroFab�, the patient cardiore-spiratory arrested. CPR was initiated and endotracheal intu-bation was performed with great difficulty due to laryngealedema. Patient was also given epinephrine and pulsereturned, although she remained hypotensive for severalhours in the Intensive Care Unit. Patient remained intubatedfor approximately 24 hours and weaned off ventilatorwithout difficulty. She received methylprednisolone,diphenhydramine, and ranitidine drip. The patient was dis-charged after 48 hours, with clinical improvement and withresidual edema and ecchymosis of the left upper extremitybut without significant coagulopathy or signs of local infec-tion, and subsequent follow-up revealed no sequelae.

Discussion: Although serious hypersensitivity reactionsto CroFab� are exceedingly rare, this case highlights theimportance of monitoring patients very carefully in criticalcare setting when giving these type products.

Conclusion: The administration of antivenom isimportant for saving lives and limbs with an acceptably lowrisk.

References

Schaeffer, T.H., Khatri, V., Reifler, L.M., Lavonas, E.J. 2012.Incidence of Immediate Hypersensitivity Reaction andSerum Sickness Following Administration of CrotalidaePolyvalent Immune Fab Antivenom: A Meta-analysis. AcadEmerg Med. 2012 Feb;19(2):121-31 Retrieved from http://www.ncbi.nlm.nih.gov/pubmed/22320362.

Keywords: Antivenom, hypersensitivity reaction, envenomation10.1016/j.toxicon.2012.04.252

252. Coral Snake Antivenin's Deadly Deadline

Robert Mannel, Olga A. Pudovka Gross, Gus A. GrossGuadalupe Regional Medical Center, Dept. of Hospitalist Medicine, Seguin,TX, USAE-mail address: [email protected] (G.A. Gross).

Background: The eastern coral snake (Micrurus fulvisfulvis) and the Texas coral snake (M. fulvius tenere) accountfor roughly 100 snakebites a year. Antivenin (M. fulvis)(Equine Origin), the only antivenom licensed in the U.S. forcoral snake bites, is no longer manufactured by Wyethpharmaceuticals. The expiration date for Lot 4030026 was10/31/2008 and has been extended multiple times to thecurrent deadline of 10/31/12. Not FDA approved andtherefore not readily available, Anticoral (Inst. ClodomiroPocado, Costa Rica) and Coralmyn (Inst. Bioclon, Mexico)have offered promising results in counteracting effects ofM. fulvius venom in rodents. Prior to coral snake antivenom,the death ratewas approximately 10% due to cardiovascularand/or respiratory failure.

Case Report: A 54-year-old female presented to theEmergency Room after having been bitten by a coral snake1 hour previously. The patient was alert and in marked


http://www.ncbi.nlm.nih.gov/pubmed/22320362

http://www.ncbi.nlm.nih.gov/pubmed/22320362



distress secondary to left ankle pain, dysphonia, and diffi-culty swallowing. The patient's past medical history waspertinent only for hypertension, with no history of snakeenvenomation or exposure to antivenom. Auscultatoryfindings and neurologic exam were within normal limitsbut she subsequently developed respiratory distress. Vitalsigns were stable. Expired Wyeth Coral Snake Antivenomwas acquired through assistance from Poison Control.Patient pre-medicated with diphenhydramine and meth-ylprednisolone. At the completion of the infusion of 5 vialsof 50 ml, she required endotracheal intubation and venti-lator support. Shortly thereafter, the patient was trans-ferred to a higher-level care facility.

Discussion: With the impending deadline approachingyet again, the already low FDA approved coral snake anti-venom availability is soon to run out. Patients will need tobe intubated and ventilated while the toxin wears off,leading to increased morbidity and mortality.

Conclusion:While coral snake bites are rare, antivenomimplementation is necessary for the patient's safety, as wellas cost-minimization.

References

Patient's medical history.Norris, R.L., 2011. Coral Snake Envenomation. Retrieved fromhttp://emedicine.medscape.com/article/771701-treatment#a1126.U.S. Food and Drug Administration 2011. Vaccines, Blood &Biologics: Expiration Date Extension for North American CoralSnake Antivenin (Micrurus fulvius) (Equine) Lot 4030026Through October 31, 2012. Retrieved from http://www.fda.gov/BiologicsBloodVaccines/SafetyAvailability/ucm155092.htm.

Keywords: Coral snake antivenom, neurotoxicity, respiratory support10.1016/j.toxicon.2012.04.253

253. Medically Significant Late Bleeding FollowingTreated Crotaline Envenomation: A Structured TopicReview

E.J. Lavonas 1,2, V. Khatri 1, C. Daugherty 3

1Rocky Mountain Poison and Drug Center, Denver Health and HospitalAuthority, Denver, Colorado, USA2Department of Emergency Medicine, University of Colorado School ofMedicine, Aurora, Colorado, USA3BTG International, West Conshohocken, Pennsylvania, USAE-mail address: [email protected] (E.J. Lavonas).

Background: Recurrent and delayed-onset thrombo-cytopenia and defibrinogenation are well-describedfollowing crotaline snakebite treated with crotalidaepolyvalent immune Fab (ovine) antivenom (FabAV).However, the likelihood that a patient will develop medi-cally significant bleeding is poorly understood.

Methods: The authors searched PubMed, Ovid MED-LINE and EMBASE from January 1, 1997 to February 6, 2012to identify all published cohort studies of patients enve-nomated by North American crotaline snakes who weretreated with FabAV. Two content experts independently

reviewed full-text articles and extracted data about studydesign, cohort, treatment, follow-up, and outcomes, whichwere then reconciled for errors and analyzed usingdescriptive statistics. In addition, case reports of bleedingthat began after initial control of the envenomationsyndrome were identified. Late bleeding was defined asbleeding that began or recurred after initial control of theenvenomation syndrome. Medically significant latebleeding was defined a priori as late bleeding that wastreated with blood transfusion, vasoactive drug infusion, orsurgery, required re-hospitalization, or was temporallyassociated with a reduction in serum hemoglobin of � 3 g/dL, reduction of hematocrit of � 8 percent, permanent ortemporary disability, or death. Summary incidence and 95%confidence interval (CI) of late and medically significantbleeding were calculated using a random-effects Poissonregression model.

Results: Eighteen unique cohort studies were identified.Five studies utilized prospective data collection. In 11studies, patients were followed actively after hospitaldischarge. A total of 901 subjects were enrolled in thesecohort studies. Of these, 8 subjects were reported to havelate bleedingwith an estimated summary incidence of 0.009(95% CI 0.003 to 0.023), including 5 subjects with medicallysignificant late bleeding, and an estimated summary inci-dence 0.006 (95% CI 0.002 to 0.019). Three patients receivedred cell transfusion; none suffered permanent sequelae. Fortwo patients, detailed clinicalmanifestations, treatment, andlaboratory test results were available for analysis. Threeadditional cases of late bleeding were identified in casereports; all were medically significant, including one death.

Conclusion: Although recurrent and delayed-onsetthrombocytopenia and defibrinogenation are commonfollowing treated crotaline snakebite, medically significantlate bleeding appears to be very uncommon.

Keywords: Recurrence, bleeding, hematologic effects, antivenom10.1016/j.toxicon.2012.04.254

254. Failure to Develop Sensitization Despite RepeatedAdministration of Ovine Fab Snake Antivenom: ASingle-Patient, Multi-Center Case Series

Eric J. Lavonas 1,2, Blaine E. Benson 3,4, Steven A. Seifert 3,51Rocky Mountain Poison and Drug Center, Denver Health and HospitalAuthority, Denver, CO, USA2Department of Emergency Medicine, University of Colorado School ofMedicine, Aurora, CO, USA3New Mexico Poison and Drug Information Center, Albuquerque, NM, USA4College of Pharmacy, University of New Mexico, Albuquerque, NM, USA5Department of Emergency Medicine, University of New Mexico School ofMedicine, Albuquerque, NM, USAE-mail address: [email protected] (E.J. Lavonas).

Background: Immediate (anaphylactic and anaphylac-toid) hypersensitivity reactions are a known complicationof antivenom therapy. Few data exist about repeatedexposure to ovine Fab antivenom in the same patient. Wereport our experience treating a 41-year oldmanwithmorethan 60 documented instances of envenomation bothnative and non-native venomous snakes over a 20-plus

http://emedicine.medscape.com/article/771701-treatment#a1126

http://emedicine.medscape.com/article/771701-treatment#a1126

http://www.fda.gov/BiologicsBloodVaccines/SafetyAvailability/ucm155092.htm






year period, including repeated administration of equineIgG, ovine Fab, and various non-native snake antivenoms.

Methods: The poison center and, when available,hospital medical records of snakebites involving thispatient were reviewed, with detailed review of those since2001.

Results: We identified 13 instances since 2001 in whichthe patient received ovine Fab antivenom (FabAV), begin-ning in an open-label clinical trial in 1993 and including twoseparate incidents treated at different hospitals over a 9-dayperiod in 2011. During the most recent treatment episodes,the patient did not receive premedication with antihista-mines or corticosteroids. During close observations, no signsor symptoms of acute hypersensitivity were observed.Because the patient was noncompliant with follow-up, dataabout serum sickness are not available. Two previousepisodes of hypotension prior to antivenom administrationmay reflect Type 1 hypersensitivity to venom.

Discussion: Sensitization to foreign proteins causingType 1 hypersensitivity reaction is a concern with repeatadministration of antivenom to the same patient. Ovine Fabantivenom is produced with a multi-step purification toremove proteins other than venom-specific Fab-fragments.However, each vial contains up to 1 gram of total protein,most of which is 50 kDa Fab fragments. Within the limi-tations of our data, it appears that this patient has notbecome sensitized despite serial exposure to FabAV, butmay have developed hypersensitivity to venom.

Conclusion: Some patients tolerate repeated adminis-tration of ovine Fab antivenom without evidence ofhypersensitivity.

Keywords: Antivenins, hypersensitivity, crotalid venoms, snake bites10.1016/j.toxicon.2012.04.255

255. Local Damage produced by Vipera and MacroviperaVenoms and Some Immunochemical Characteristics

Néstor R. Lago 1, R. de Adolfo Roodt 1, Irving Archundia 2,Daniela M. Rocco 1, Vanessa Costa de Oliveira 1,Pablo I. Regner 1, Jorge Zárate 1, Alejandro Alagón 2,Roberto P. Stock 2

1 Laboratorio de Toxinopatología, Centro de Patología Experimental yAplicada, Facultad de Medicina, Universidad de Buenos Aires, Uriburu 950,5oPiso, Lab.555 (1427) Buenos Aires, Argentina2 Instituto de Biotecnología de la Universidad Autónoma de México, MexicoE-mail address: [email protected] (R. de Adolfo Roodt).

Background: The information on the toxic activities isnot abundant, by this reason, the toxic activities of Vipera(V) aspis aspis (Vas), V. ammodytes ammodytes (Vaa),V. amodytes montandoni (Vam), V. berus (Vb), V. xhantina(Vx), Macrovipera (M.) lebetina obtusa (Mlo) andM. schweizeri (Ms) venoms were studied.

Material and Methods: The hemorrhagic, necrotizingand inflammatory-edematogenic activities were deter-mined in rats (Wistar). We also performed a histologicalstudy of lesions in animals injected intramuscularly(extensor digitorum longus) with sub-lethal doses of thevenoms. The reactivity with homologous and heterologousantivenoms was studied.

Results: All venoms exhibited some hemorrhagicactivity, the most hemorrhagic being Vb (w 12 MHD/mg)and Mlo (w 6 MHD/mg). Those with lowest hemorrhagicactivity were the venoms of Vama (w 2.5 MHD/mg) andVamm (w1.6 MHD/mg). The venoms of Vb, Vx, Vas, Mlo andMs venoms produced skin necrosis (studied as MND).Inflammation – edema was observed in all venoms indifferent degrees. The venom with the lowest inflamma-tory activity was Vx (comparable to the controls) and thosewith the highest activity were Ms and Vb (two fold overcontrols). No activity on hemostasis (plasma and fibrin-ogen) was observed. In all cases venoms were somewhatmyotoxic. After 24 h, rats injected intramuscularly weresacrificed and the injected muscle and contralateralcontrol (injected with the same volume of NaCl 0.15 M)were processed for optical microscopy and stained with H& E. Venom of Mlo caused acute inflammation, necrosisand hemorrhage of intermediate magnitude whereas thatof Ms showed lower acute inflammation, hemorrhage andnecrosis. Vaa caused important acute inflammation,edema and hemorrhage and necrosis while that of Vamcaused necrosis and acute inflammation but low edemaand hemorrhage. Vb venom caused hemorrhage, edemaand acute inflammation but necrosis was lower. Vasvenom caused acute inflammation, edema and necrosisbut intermediate hemorrhage and that of Vx causedconsiderable edema and hemorrhage but intermediatenecrosis. All venoms cross-reacted in vitro with anti-Vipera, anti-Bothrops and anti-Crotalus durissus terrificusantivenoms.

Discussion: In some cases the effect observed inmuscles could not be detected by the common assays fordetermining hemorrhage and necrosis. Local lesions causedby the venoms were no correlated with their systemictoxicity (lethal dose). Although intramuscular injection isnot the most common inoculation route in viper enveno-mation, the possibility of muscular lesions should beconsidered in bites by these species, since in all the caseslocal damage was observed.

Keywords: Vipera, Macrovipera, myotoxicity, venom, snakes10.1016/j.toxicon.2012.04.256

256. A Rapid Reconstitution Method for CroFab�Polyvalent Immune Fab (ovine)

David Gerring, Richard Branton, Terry Prime,Thomas R. King, Emmanuel M. MahlisBTG International Inc., West Conshohocken, PA, USA and London, UKE-mail address: [email protected] (T.R. King).

Background: Reconstitution of CroFab� lyophilizeddrug product is performed according to its currentapproved US labeling, which requires the use of 10 mLsterile water for injection followed by up to a minimum of36 minutes of gentle swirling of the vial. CroFab� has beenclinically demonstrated to be most effective when admin-istered within 6 hours of snake envenomation, andimproved clinical outcomes are correlated with quickertiming of administration.




Methods: An analytical study was designed to comparethe physicochemical properties of 3 separate batches ofCroFab� when reconstituted using the standard procedure(10 mL WFI with gentle swirling) and a modified rapidprocedure using 18 mL 0.9% saline and manual inversion.The physical and chemical characteristics of the same 3batches were assessed using various analytic methodolo-gies associated with routine quality control release testing.In addition further analytical methodologies were appliedin order to elucidate possible structural changes that maybe induced by the changed reconstitution procedure.

Results: Batches A, B, and C required mean reconstitu-tion times of 25 min 51 secs using the label method and 3mins 07 secs (88.0% mean decrease) using the modifiedmethod. Physicochemical characteristics (color and clarity,pH, purity, protein content, potency) were found to behighly comparable. Characterization assays (dynamic lightscattering, analytical ultracentrifugation, LC-MS, SDS-PAGEand circular dichroism spectroscopy were also all found tobe comparable between methods.

Discussion:When comparing CroFab� batches that werereconstituted using the labeled and modified methods, thephysicochemical and biological (potency) characteristics ofCroFab�were not significantly changedwhen challenged bythe various standard analytical methodologies applied inroutine quality control analysis. Additionally, no changes inthe CroFab� molecule regarding degradation, aggregation,purity, structure, or mass were observed.

Conclusion: The analyses performed support the use ofthe more rapid reconstitution method using 18 mL 0.9%saline in order to allow a significantly reduced time toadministration of CroFab� to patients in need.

Keywords: Antivenoms, polyvalent immune fab, crotalid envenomation10.1016/j.toxicon.2012.04.257

O. Spiders

257. Comparative Analysis of Transcriptomes ofPhoneutria pertyi and P. nigriventer Venom Glands

Marcelo R.V. Diniz 1, Camilla R.L. Machado 1,Mauricio A. Mudado 2, Ana Luiza B. Paiva 1

1Centro de Pesquisa e Desenvolvimento Carlos Ribeiro Diniz, FundaçãoEzequiel Dias, Belo Horizonte, Brazil2 Empresa Brasileira de Pesquisa Agropecuaria – Embrapa, São Carlos, BrazilE-mail address: [email protected] (M.R.V. Diniz).

Background: Species of the genus Phoneutria known as"aranha-armadeira" or armed spiders are responsible fora large number of spider bites in Brazil. Until recently, allstudies on the venom of the genus have been restricted tothe species P. nigriventer. However, some recent proteomicstudies revealed that other species venoms also containsa wide variety of proteins and peptides, including neuro-toxins which act on the ion channels and chemical recep-tors of the neuro-muscular systems of insects andmammals. Thus, these venoms have emerged as invaluabletools for research, drug discovery and drug developmentwith application in medicine and agriculture.

Methods: In order to find novel venom componentswith biological activity and to provide a database forcomparative study with the previously described P. nig-riventer venom gland transcriptome, we constructeda plasmidial cDNA library from the species Phoneutria pertyimRNA venom gland to generate Expressed Sequence Tags(ESTs) data.

Results: After editing, 710 good quality reads wereclustered and 295 unique sequences were obtained (106contigs and 189 singlets). Of these, 197 (67%) had a highdegree of homology to spiders toxins deposited in theUniprot database, most are P.nigriventer toxins isoforms.We observed that P. pertyi venomgland transcriptomeweremore abundant in insecticidal toxin sequences thanP. nigriventer one. We also found new sequences for puta-tive toxins in P. pertyi transcriptome, indicating that theycan be novel toxins.

Conclusions: These results show that although thesespider venoms contain a similar range of toxins isoforms,the expression levels of each type of toxins are different,and also they contain toxins with unique sequences, whatcan suggest adaptation to different environments.

Keywords: Phoneutria venom gland, transcriptome, neurotoxin10.1016/j.toxicon.2012.04.258

258. Unusual Cases of the Spider CheiracanthiumPunctorium Biting in Volgograd Region, Russia

Vasiliy I. Emtsov 1, Yury N. Ostapenko 2, Sergey S. Larionov 1

1Volgograd Regional Poisoning Treatment Centre, Volgograd RegionalNarcological Hospital, Volgograd, Russian Federation2Research and Applied Toxicology Centre, Federal Medical and BiologicalAgency, Moscow, Russian FederationE-mail address: [email protected] (Y.N. Ostapenko).

Background: Beginning in 2009 cases of bites by thespider Cheiracanthium punctorium, were reported in theVolgograd region of Russia. There had been no previousreports of bites by this species in that geographic area. Theaim of this study was characterization of the epidemiologyand clinical picture of such envenomations.

Methods: Retrospective analysis of 19 case reports of in-patients in Volgograd Regional Poisoning Treatment Centerin 2010-2011. Only cases of confirmed bites by Cheir-acanthium punctorium were included. Identification of thespider was made by qualified specialists in the VolgogradState University.

Results: Cases of biting of poisonous animals in Russiaare responsible for 1,9% of all poisoning cases in patientsadmitted to toxicological treatment centers. According todata from 29 centers in 2010 549 (43,4%) patients wereadmitted with snake bites, 693 (54,8%) with poisonousinsect and spider bites, and 23 (1,8%) after contact withpoisonous sea animals. Bites of the common viper, ViperaBerus, occur over the entire country. In addition, in territoriesbordering Central Asian countries, including Volgogradregion, the bites by tarantulas and black widow spiders areregularly reported. The number of patients admitted to theVolgograd Regional Poisoning Treatment Centre withpoisonous spider biting for the last two years was 111,




including 19 with Cheiracanthium punctorium. During thefirst minutes after the bite by C. punctorium, 90% of patientsreported stabbing and burning pain, with 100% reportingreddening and swelling at the site of the bite. Systemiceffects were noted in 70 % of cases, manifested by nausea,vertigo, and general weakness. Pain elsewhere in the bodyoccurred in 30%, including low back pain, extremity pain,and headache. Allergic reactions, manifested as shock andQuincke's edema,was seen in one patient, with urticaria andallergic dermatitis seen in 2 patients. Twelve patientsdemonstrated insignificant leukocytosis with increasednumbers of neutrophils and eosinophils, aswell as increasedtransaminases and amylase in two patients. Therapy wassymptomatic and included non-opioid analgesics, antihis-tamines, and corticosteroids as indicated. Treatment wascontinued in mild cases for an average hospital stay of 4days, and for 7 to 9 days in more serious case. Outcomes inall cases were favorablewith no skin ulceration and no long-term sequelae.

Discussion: Human bites by the spider Cheiracanthiumpunctorium are known in Europe and the Americas,whereas its appearance in Russia has not previously beenreported. One possible explanation is anomalously hotweather during the summer months for the past 2 years.The clinical picture demonstrated by patients in our regioncorresponds to those in the published literature.

Conclusion: Poisoning by Cheiracanthium punctoriumvenom could be topical not only for Volgograd region, butalso other regions of Russia, if anomalously hot summertemperatures continue to occur. There does not appear tobe a significant risk of death and the management issymptomatic and supportive.

Keywords: Cheiracanthium Punctorium poisoning10.1016/j.toxicon.2012.04.259

259. Molecular Basis for the Interaction of TarantulaToxin Jingzhaotoxin-III (b-TRTX-Cj1a) with the VoltageSensor of Kv2.1 Channel

Huai Tao, Jinjun Chen, Yucheng Xiao, Zhonghua Liu,Songping LiangCollege of Life Sciences, Hunan Normal University, Changsha, Hunan, ChinaE-mail address: [email protected] (S. Liang).

Background: Animal venoms contain a fascinatingarray of diverge peptide toxins that have cross-activities ofdifferent types of voltage-gated ion channels. However, theunderlying mechanism remains unknown. Jingzhaotoxin-III (JZTX-III), a 36-residue peptide from the tarantula Chi-lobrachys jingzhao, can selectively inhibit both Nav1.5 andKv2.1 channels without affecting the majority of other ionchannel subtypes. JZTX-III could trap Nav1.5 DII voltagesensor at closed state by binding to DIIS3-S4 linker.

Results: In this study, we report that JZTX-III docked onKv2.1 voltage sensor paddle. Ala-mutation of single residueof Phe274, Lys280, Ser281, Leu283, Gln284 or Val288 canreduce the effects of JZTX-III evidently. Among them, S281is the most crucial residue and the substitution with Ala,Phe, Ile, Val or Glu increased the IC50 value by over 34-folds.

Discussion: Interestingly, the bioactive surfaces of JZTX-III interacting with Kv2.1 and Nav1.5 are very different fromeach other. Although a hydrophobic patch formed by Trp8,Trp28 and Trp30 is important for JZTX-III interaction withboth channels, the patch conserved among other inhibitorsof Kv2.1 has been shown to play a critical role in parti-tioning into lipid membrane. While three residues Asp1,Glu3 and Trp9 are critical only for inhibition of Nav1.5,three charged residues (Arg13, Lys15, and Glu34) and onehydrophobic residue (Val33) are only crucially involved ininteraction with Kv2.1.

Conclusions: These results strongly supported thatanimal toxins might use different bioactive surface totarget the voltage sensor paddles of different ion channels.Our findings provided new molecular insights for thedesign of more specific and more potent ion channelinhibitors.

Keywords: Spider toxin; voltage-gated ion channel; ion channel inhibitors10.1016/j.toxicon.2012.04.260

260. Hainantoxin-III Inhibits Voltage-Gated SodiumChannel Nav 1.7 and Extenuates Inflammatory Pain inAnimal Models

Zhonghua Liu 1, Qi Zhu 2, Tianfu Cai 1, Ze Wu 3, Jing Li 1,Dan Li 1, Weiwen Ning 1, Yu Liu 3, Meichun Deng 1,Weijun Hu 1, Songping Liang 1

1College of Life Sciences, Hunan Normal University, Changsha, Hunan, China2 LC Sciences, Houston, TX, USA3College of Chemistry and Chemical Engineering, Hunan Institute of Scienceand Technology, Yueyang, Hunan, ChinaE-mail addresses: [email protected] (Z. Liu), [email protected](S. Liang).

Background: Voltage-gated sodium channels (VGSCs)are important for nociceptive signaling and are potentialtargets for treating pain. Among nine VGSCs subtypes,Nav1.7 is preferentially expressed in dorsal root ganglion(DRG) and sympathetic ganglion neurons. The importantroles Nav1.7 plays in inflammatory pain have been rela-tively well established.

Results: Here, we demonstrated that hainantoxin-III(HNTX-III), purified from the venom of spider O.hainana, could selectively inhibit tetrodotoxin-sensitive(TTX-S) in rat DRG cells. Hainantoxin-III is a 33-residuepolypeptide with three disulfide bridges and adoptsinhibitor cystine knot (ICK) motif, imparting it extraor-dinary stability. Hainantoxin-III was able to inhibithNav1.7 expressed in HEK 293 cells through a novelmechanism identical to huwentoxin-IV that couldinhibit Nav1.7 by trapping DII S4 voltage sensor on theclosed state. Furthermore, nanomolar HNTX-III adminis-trated by intramuscular injection could extenuateinflammatory pain on formalin test and abdominalconstriction (writhing) test. Its analgesic effect wouldcompare favorably with that of morphine and wasn'tassociated with motor side effects even at 20-folderhigher dose.

Conclusion: HNTX-III would be a novel probe useful inthe investigating TTX-S VGSCs including Nav1.7 and an





interesting lead that may be helpful in designing noveldrugs for pain treatment.

Keywords: VGSCs, Nav1.7, spider, hainantoxin-III, antinociceptive10.1016/j.toxicon.2012.04.261

261. Expanding Structural and Functional Diversity ofSpider Venom Compounds

Alexander A. Vassilevski, Eugene V. GrishinM.M. Shemyakin and Yu.A. Ovchinnikov Institute of Bioorganic Chemistry,Russian Academy of Sciences, Moscow, Russian FederationE-mail address: [email protected] (A.A. Vassilevski).

Background: Spiders are masters of venom production.The diversity of both the chemistry and pharmacology ofspider venom compounds is striking. In single spidervenom, several hundred different components may beidentified, varying from inorganic salts to high-molecular-mass proteins. These components aim at a large number oftargets in the prey/aggressor organism.

Results and Discussion:We shall summarize our recentprogress in the field. (i) It becomes obvious that spidervenom peptides not only hit the "classical" targets, the vitalcomponents of the nervous signaling mechanisms, but alsoaffect many "non-classical" targets, such as sensoryreceptors. The recently identified purotoxins modulatingmammalian purinergic receptors present prospectiveanalgesic drug leads. (ii) The combinatorial libraries oftoxins found in spider venom generate novel modes ofaction. A good example is the family of insect-selective b/d-agatoxins from the spider Agelena orientalis. These toxinspossess the ability to modulate both the activation andinactivation processes in insect sodium channels, resem-bling both a- and b-toxins of scorpions. (iii) "Traditional"peptide toxins from spider venom show neurotoxicity. Itseems that evolution created equally effective cytolyticpeptides. The "cytolytic champion" spider Lachesana tar-abaevi produces a host of linear peptides forming a dozenof families. Their activity screening resulted in identifica-tion of antimicrobial substances exhibiting potent anti-Chlamydia effects. (iv) A new level of complexity is intro-duced by the so-called "modular" toxins. They combine intheir structure two modules, or domains, each corre-sponding to a "usual" spider toxin. We describe severalmodular toxins with different domain arrangement.

Conclusion: The diversity of spider venom compoundsis ever-expanding and further research is greatlyanticipated.

Keywords: Spider venom, neurotoxin, cytolytic peptide10.1016/j.toxicon.2012.04.262

262. Spider Venom Components Affecting the Functionof Purinergic Receptors

Eugene GrishinShemyakin and Ovchinnikov Institute of Bioorganic Chemistry, RussianAcademy of Sciences, 16/10 Miklukho-Maklaya, Moscow, Russian FederationE-mail address: [email protected].

Review: Purinergic P2X receptors are membrane ionchannels that open in response to the binding of extracel-lular ATP. These receptors can be involved in pain mecha-nisms and thereby constitute possible targets for analgesicdrugs. Until recently no natural venom or its individualcomponents were known to affect P2X receptors. The firstnatural peptide, which exerts selective inhibitory action onP2X3 receptors, was purotoxin-1 (PT1) isolated from thevenom of the Central Asian spiderGeolycosa sp. PT1 contains35 amino acid residues with four intramolecular disulfidebridges. A novel polypeptide named purotoxin-2 (PT2) fromthe same spider venom consists of 64 amino acid residueswith four disulfide bonds and features C-terminal amida-tion. PT1 and PT2 produce inhibitory effects on P2X3-mediated currents via stabilization of the desensitized stateof the P2X3 receptor. Six structural homologues of PT1 werefound in EST databases generated from venom glands ofvarious spiders. For functional studies, six recombinantanalogs of PT1 from different spiders were produced. PT1analogs were studied on P2X2, P2X3, and P2X2/3 receptorsin rat DRG neurons. Out of six PT1 analogs two peptideswere inactive. It was found that one recombinant peptideinhibited P2X2-generated currents, three analogs showedstable inhibitory action on P2X3 receptors, and one of themalso produced a small potentiating effect on the currentgenerated by P2X2/3 receptors. So, spider venoms containa family of polypeptides possessing powerful and selectiveinhibitory action on P2X receptors. Some of these poly-peptides demonstrate marked antinociceptive activity.

Keywords: P2X receptors, spider venom, purotoxins10.1016/j.toxicon.2012.04.263

263. Molecular Cloning and Characterization ofa Sphingomyelinase D from Loxosceles adelaida,a Brazilian Brown Spider from Karstic Areas

Giselle Pidde-Queiroz 1, Rute M. Gonçalves-de-Andrade 1,Cinthya K. Okamoto 1, Tiago J. Sobreira 2,Paulo S.L. de Oliveira 2, Mário T. Murakami 2,Carmen van den Berg 3,1, Denise V. Tambourgi 11 Immunochemistry Laboratory, Butantan Institute, São Paulo, Brazil2National Laboratory for Biosciences, National Centre for Research in Energyand Materials, Campinas, Brazil3Department of Pharmacology, Oncology and Radiology, School of Medicine,Cardiff University, Cardiff, UKE-mail address: [email protected] (D.V. Tambourgi).

Background: Envenomation by spiders belonging to thegenus Loxosceles, found in temperate and tropical regions ofAmerica, Africa and Europe, commonly results in impressivelocal necrotic skin lesions and can also cause systemiceffects, including intravascular hemolysis. Loxosceles is themost poisonous spider in Brazil and three different synan-thropic species of medical importance are known, i.e., L.intermedia, L. gaucho, L. laeta, and more than 6000 cases ofenvenomation by L. intermedia alone are reported each year.We have purified, characterized, cloned and expressed thetoxins from L. intermedia and L. laeta venom that areresponsible for all the local and systemic effects induced bythe whole venom. Highly homologous proteins with





sphingomyelinase activity (SMase D) were able to induce allthe local and systemic effects induced by whole venom.Loxosceles species are present in several different habitats,including the karstic environment, and in Brazil it is themost common troglophile arachnid. The aims of the presentstudy were to clone and express SMases D from the spidergland of L. adelaida, captured in the caves of PETAR (Brazil),to compare the functional activities of the recombinantproteins with toxins from synanthropic species and toinvestigate the inter- and intra-species cross-reactivities ofantibodies raised against the purified recombinant proteins.

Methods: The gene was amplified by RT-PCR and clonedinto a prokaryotic expression vector pRSETA. The recombi-nant protein was compared with the previously character-ized SMases D from synanthropic spiders, in terms of theirbiological, biochemical and structural properties.

Results and Discussion: The L. adelaida SMase D A1cDNA exhibits similarity with previously characterizedSMases D. Antiserum produced against the recombinantSMases D from L. intermedia and from L. laeta was highlycross-reactive against L. adelaida SMase D A1, and alsoexhibit a high level of recognition to SMases present in L.adelaida and L. gaucho venoms. SMase DA1 is able to inducea typical dermonecrotic reaction and transform erythro-cytes into activators of the autologous complement system,but with lower toxic potential than the SMases D fromsynanthropic species. Based on sequence and structuralsimilarities, the SMases D can be grouped into two classesdepending on the presence of an additional disulphidebridge between the catalytic loop and flexible. L. adelaidaSMase DA1 is a class II member and conserves all structuralfeatures for catalytic activity upon sphingomyelin.

Financial support: FAPESP, CNPq and INCTTOX

Keywords: Loxosceles adelaida, venom, sphingomyelinase D10.1016/j.toxicon.2012.04.264

264. Milking and Partial Characterization ofPamphobeteus spp (Aranae; Theraphosidae) Venom,from the Colombian Andean Region

Sebastian Estrada Gomez 1,2, Leidy Vargas Munoz 1,Alejandro Ramirez 1, Juan Quintana Castillo 1

1Ophidism Research Program, University of Antioquia, Medellín,Colombia, USA2Assistant profesor, Pharmacy Faculty, University of Antioquia, Medellín,Colombia, USAE-mail address: [email protected] (S.E. Gomez).

Background: In this work we report the first studiesof milking and partial characterization of the Pampho-beteus venom made in Colombia using techniques asindirect hemolytic and coagulant activity, electropho-resis, HPLC and peptides identifications. Different reportsshows that this venoms are a rich mixture of salts,nucleotides, free amino acids, neurotransmitters,peptides, proteins and enzymes affecting invertebratesand vertebrates. They affect mainly the central nervoussystem due the content of neurotoxins affecting ionicchannels at pre and postsynaptic level and neurotrans-mitter exocytosis.

Methods: The specimens used in this study come fromthe Andean region of Colombia (Antioquia province) andwhere maintained in captivity with food and water adlibitum. 12% PAGE-SDS electrophoretic gels were usedruned at 150V for 60minutes. A HPLC reverse phase system(RP-HPLC) with a C-18 column was used to obtain thechromatographic profile. N-terminals were obtained usinga HPLC-nESI-LC/MS system. For partial biochemical char-acterization, hemolytic (egg yolk method) and coagulantactivity were used according with Gutierrez et al andTheakston and Reid respectively.

Results: After each sider milking, a low viscosity,colorless, with a density of 1.01 mg/ml and a pH of 5 wasobtained. In all cases female quantity of venom obtainedwere larger regarding male venom quantity (20% wetweigh and 37% dry weigh lager). The humidity percentagedid not show a significance difference between males andfemales (20.45�1.68 %). The electrophoretic profileshowed the presence of low molecular compounds (5-15kDa range) that usually corresponds to peptides and someneurotoxins. In the chromatographic profile we obtainedwell-defined fractions that allowed a posterior identifi-cation of its N-terminal through a HPLC-nESI-LC/MSsystem. After a BLAST, we found that this fraction hasa high homology with ESTx neurotoxins (Eurypelma-spider-toxins) that affect mainly calcium channels andwith Lychasmucronatus scorpion genus neurotoxins thataffect mainly potassium channels. The biochemical assaysindicates that this venoms exhibit a catalytic activitymore over the neurotoxic activity based the proteinssimilarity.

Discussion: The Colombian Pamphobeteus spidervenom shows general content similarities regarding othertheraphosinae spiders. The high amount of venom yieldedby females may be related with sexual dimorphismexpressed in this species. Non-expected catalytic andcoagulant activities were showed in this venom.

Conclusion: The BLAS indicates that this venom hasa neurotoxic effect mediated through ionic channels. Thecatalytic activity could indicate that spiders uses thevenom not only to paralyze they pry but for predigest it aswell.


Table

Physicochemical properties Result

Color ColorlessDensity 1.01 mg/mlpH 5


Keywords: Pamphobeteus, neurotoxic, catalytic10.1016/j.toxicon.2012.04.265

Solubility in aqueous solvents 100%

265. Poke but Don't Pinch: Risk Assessment andDefensive Behaviors of the Western Widow Spider(Latrodectus hesperus)

David R. Nelsen, Allen M. Cooper, Gerad A. Fox,Wayne Kelln, William K. HayesLoma Linda University, Department of Earth and Biological Sciences, LomaLinda, California, USAE-mail address: [email protected] (D.R. Nelsen).

Background: The defensive behaviors of the WesternWidow Spider (Latrodectus hesperus), a medically relevant,

synanthopic species found throughout western NorthAmerica, are poorly understood.

Methods: To elicit defensive behaviors, wild-caughtadult females (N ¼ 43) were either prodded once (lowthreat), prodded repeatedly (medium threat), or pinchedrepeatedly (high threat) with gelatin “fingers” ina repeated measures design. We collected data on defen-sive behaviors that were non-aggressive (retractinglimb(s), fleeing, playing dead) and aggressive (silk flicking,biting).

Results: The proportion of spiders whose first responseto provocation was non-aggressive was 100% across allthreat levels. Besides flight, the most common defensivebehavior elicited in low, medium, and high threat condi-tions was retraction (16%), silk flicking (56%), and biting(59%), respectively. Spiders were significantly more likelyto bite (p<.0001) “fingers” when pinched (59%) than whenprodded singly (0%) or repeatedly (2%). Number of bitesdelivered (mean þ/- SD) was significantly greater forpinched (2.7 þ/- 3.6) than repeatedly prodded (0.02 þ/-0.15) spiders. Spiders were significantly more likely to flicksilk at fingers when pinched (43%) or prodded repeatedly(56%) than when prodded singly (5%). Number of silk flickswas also significantly greater for pinched (1.8 þ/- 2.9) andrepeatedly prodded (3.7 þ/- 5.5) than for singly prodded(0.05 þ/- 0.21) spiders.

Conclusions: We conclude that L. hesperus is first non-aggressive, but if compressed between two object will bitedefensively. This helps to confirm the idea that theWesternWidow Spider is generally non-aggressive. Additionalresults from studies underwaywill be presented, including,but not limited to, defensive venom metering and onto-genetic development of defensive behaviors.

Keywords: Latrodectus hesperus, defensive behaviors, risk assessment,ontogeny10.1016/j.toxicon.2012.04.266

266. C5a Receptor is Cleaved by MetalloproteasesInduced by Sphingomyleinase D in Loxosceles SpiderVenom

Carmen W. van den Berg 1,2,Rute Maria Gonçalves-de-Andrade 2, Cinthya K. Okamoto 2,Denise V. Tambourgi 21Department of Pharmacology, Oncology and Radiology, School of Medicine,Cardiff University, Cardiff, UK2 Immunochemistry Laboratory, Butantan Institute, São Paulo, BrazilE-mail address: [email protected] (C.W. van den Berg).

Background: Neutrophils are involved in numerouspathologies and are considered to be one of the mainfactors contributing to the establishment of cutaneousloxoscelism after envenomation by the Loxosceles spider.Neutrophils are attracted to the site of envenomation bylocally generated C5a and contribute to the tissuedestruction. We have investigated the effects of this spidervenom on the receptor for C5a: C5aR/CD88, a seventransmembrane G-protein coupled receptor.

Results: We show here that the Loxosceles venominduces the cleavage of the C5aR at two sites, resulting in




the release of the extracellular N-terminus, whileretaining part of the transmembrane regions. Usingspecific inhibitors it was shown that the cleavage wasinduced by activation of an endogenous metal-loproteinase of the adamalysin (ADAM) family, whichwas activated by the sphingomyelinase D in the venom.Although it resulted in the near complete loss of the N-terminus, C5a was still able to interact with the C5aR andinduce a small increase in intracellular calcium and thesecretion of IL-8.

Discussion: The cleavage of the C5aR may well bea protective response after envenomation, rather thancontributing to the pathology of Loxosceles envenomationand may represent a general mechanism of how the bodyprotects itself against excess C5a generation in circum-stances such as sepsis. Supported by FAPESP and CNPq.

Keywords: C5a receptor, Loxosceles spider venom, adamalysin, sphingo-myelinase D, dermonecrosis10.1016/j.toxicon.2012.04.267

267. A Survey of Venom from the Spitting SpiderScytodes includes Novel Toxins

Pamela A. Zobel-Thropp 1, Sandra Correa 2, Jessica Garb 2,Greta Binford 1

1 Lewis & Clark College, Dept. of Biology, Portland, OR, USA2University of Massachusetts, Dept. of Biological Sciences, Lowell, MA, USAE-mail address: [email protected] (P.A. Zobel-Thropp).

Review: Spiders in the family Scytodidae are foundworldwide, include hundreds of species and are known fortheir unique prey capturing technique – spitting a zig-zagof glue to tether prey to a surface. The effectiveness of thissticky mixture is based on a combination of contractionand adhesion thereby immobilizing prey long enough thatthe spider can envenomate. We are studying the “ven-ome” of Scytodes thoracicawith the goals of describing thecomposition of chemicals involved in this unusual systemof prey capture, discovering new toxins and betterunderstanding the phylogenetic breadth of toxins knownin other Haplogynes. To do this we have constructed cDNAlibraries from venom glands and sequenced over 800transcripts from this species. We are comparing thetranscriptome with the venom proteome of the sameindividuals using MudPIT analyses of proteins in venomand glue. A comparison of the transcriptomic and pro-teomic components helps confirm transcripts that codefor extruded venom components. We have identifiedtranscripts homologous to astacin metalloproteases, andpeptides with significant similarity to U1-agatoxin-Ao1afrom Agelena orientalis, U12-lycotoxins from Lycosa sin-goriensis and U1-nemetoxin-Csp1a from Calisoga sp. Inaddition, we have identified a highly expressed glycinerich peptide that constitutes about 50% of the venomgland transcripts. We have ascertained 13 distinct groupsof candidate peptide toxins, each containing signalsequences recognized as sites for cleavage and processingof mature toxins and patterns of cysteine scaffoldingmotifs characteristic of insecticidal neurotoxins found inspider venoms studied to date. The presence of astacins

suggests that the presence of this toxin predates the mostrecent common ancestor of the superfamily Scytodoidae,while the lack of sphingomyelinase D transcripts isconsistent with hypotheses that this medically relevanttoxin is unique to sicariids.

Keywords: Venom, transcriptome, proteome, Scytodidae10.1016/j.toxicon.2012.04.268

268. Biological activities of PnTx2-6, an exciting toxinfrom spider Phoneutria nigriventer

Maria Elena de Lima 1, Carolina N. Silva 1,Juliana S. Cassoli 1,3, Fernanda S. Torres 1, Marcelo R. Diniz 2,Márcia H. Borges 2, Marta N. Cordeiro 2,Adriano M.C. Pimenta 1, Kênia N. Pedrosa 4,Steve Peigneur 3, Jan Tytgat 31 Lab. Venenos e Toxinas Animais, Depto. Bioquímica e Imunologia,ICB -Universidade Federal de Minas Gerais, Brazil2Centro de Pesquisa, Fundação Ezequiel Dias, Belo Horizonte, MG, Brazil3 Laboratory of Toxicology, University of Leuven (KUL), Leuven, Belgium4 School of Medicine, – Georgia Health Sciences University, Medical College ofGeorgia, Augusta/GA, USAE-mail address: [email protected] (M. Elena de Lima).

Background: we previously have shown that PnTx2-6,a peptide toxin (MW 5.291.3Da) purified from the venomof the armed spider P. nigriventer potentiates erectionfunction in normotensive rats and also restores theerectile function in hypertensive (DOCA Salt) indiabetic and old rats. These effects are nitric oxide (NO)-mediated. The results presented here show the action ofPnTx2-6 in glutamate release from synaptosomes of ratbrain and upon ion channels expressed in Xenopus laevisoocytes.

Methods: synaptosomes were prepared from rat braincortices and incubated in the presence or absence ofPnTx2-6. The glutamate (L-glu) release was measuredafter reaction with NADPþ in the presence of the enzymeglutamate dehydrogenase, by a fluorimetric method. Theactivity on ion channels was assessed by electrophysio-logical measurements using the two electrode voltage-clamp technique on cloned voltage-gated sodium andpotassium channel subtypes, expressed in Xenopus laevisoocytes.

Results: PnTx2-6 increases the glutamate release fromsynaptosomes in a time- and dose- dependent manner(EC50 ¼ 20nM). The toxin obtained by heterologousexpression in E.coli also reproduces the same effect. Theincrease in L-glu release was totally inhibited by tetro-dotoxin and only partially inhibited by EGTA and BAPTA.Calcium channels blockers, such as Ѡ conotoxin MVIICand GVI A, inhibited only partially L-glu released byPnTx2-6, although nifedipine did not show any inhibi-tion. The toxin, among others, slowed down the inacti-vation and blocked of sodium currents, with differentpotency for the different isoforms of sodium channels,being more active on Nav1.3 (EC50 ¼ 55nM). However,PnTx2-6 did not show any effect upon Kþ channels(Kv1.5, Kv2.1 and Kv3.1).

Discussion: Our previous results suggest that thepotentiating effect of PnTx2-6 on erectile function may be




mediated by sodium channels. Here we show that thistoxin releases L-glu from synaptosomes of rat brain and hasa wide range of action on sodium channels expressed inX. laevis oocytes.

Conclusion: PnTx2-6 seems to exert its primaryaction through sodium channels acting on a wide range ofthem. This toxin, besides potentiating erectile function,releases L-glu from rat brain synaptosomes. We don't knowyet if the release of L-glu and the potentiation of erectilefunction can be correlated.

Financial support: The research agencies from Brazil:CNPq, CAPES and FAPEMIG

Keywords: Spider toxin, PnTX2-6, sodium channels10.1016/j.toxicon.2012.04.269

269. Understanding the Chemical Diversity andEvolution of Spider Venoms using ComparativeTranscriptomics

Sandy S. Pineda 1, Emily S. Wong 1,3, Bryan G. Fry 2,Greta J. Binford 2, Glenn F. King 1

1 Institute for Molecular Bioscience, The University of Queensland, Brisbane,Australia2 School of Biological Sciences, The University of Queensland, Brisbane,Australia3Department of Biology, Lewis & Clark College, Portland, OR, USAE-mail address: [email protected] (G.F. King).

Background: Spiders are the most speciosevenomous animal and along with predatory beetles arethe most successful terrestrial predators, with over42,000 extant species described to date. Their evolu-tionary success is due in large part to the production ofa highly complex venom that is used to subdue preyand deter predators. Recent mass spectrometric anal-yses indicate that the venoms of some spiders contain>500 different peptides but very little is known abouthow these arachnids evolved such complex chemicalcocktails.

Methods: We sampled venom-gland transcriptomesfrom a taxonomically diverse group of spiders chosen toprovide reasonable coverage of the Araneae phylogenetictree. 454 sequencing using the Roche GS-FLX platform waschosen to provide a good compromise between depth ofcoverage and accuracy. Our panel included three spiders ofmedical importance.

Results: The resulting 454 datasets have provided anunprecedented view of the molecular complexity ofspider venoms, with >35 different protein superfamiliesidentified to date. Many of these toxin superfamiliesencode 3D scaffolds not previously identified in spidervenoms. Comparison of the venom-gland transcriptomeshas provided us with the first ever (albeit crude) map ofthe evolutionary pathway of spider toxins and it hasenabled us to identify evolutionarily conserved toxinfamilies.

Keywords: Spider venom, venom proteome, venom evolution, venompeptides, comparative transcriptomics, bioinformatics10.1016/j.toxicon.2012.04.270

270. Site-Directed Mutagenesis of rPnTx2-6 - ARecombinant Phoneutria nigriventer Spider VenomToxin – Reveals Important Amino Acids for ItsPharmacological Activities

Marcelo R.V. Diniz 1, Maria Elena Lima 2,Fernanda S. Torres 2

1Centro de Pesquisa e Desenvolvimento Carlos Ribeiro Diniz, FundaçãoEzequiel Dias, Belo Horizonte, Brazil2Departamento de Bioquímica e Imunologia, Instituto de Ciências Biológicas,Universidade Federal de Minas Gerais, Belo Horizonte, BrazilE-mail address: [email protected] (M.R.V. Diniz).

Background: PnTx2-6 toxin of the Phoneutria nig-riventer spider venom was cloned and expressed as thio-redoxin fusion protein in the cytoplasm of Escherichia coli,cleaved from the conjugate and purified by HPLC [Torres etal. (2010) Toxicon 56, 1172–1180]. As does the native toxinpurified from the spider venom, the recombinant toxin -rPnTx2-6 - potentiates erectile function when injected intothe rat, and causes glutamate release from rat corticalsynaptosomes.

Methods: The cloned gene was used to make sitedirectedmutations, to attempt to pinpoint the residues thatare involved in producing this effect.

Results: Six mutant genes were obtained and expressedwith specific amino acid substitutions. Five were furtherpurified and their molecular masses were checked. Themutant proteins were tested, and compared with those ofthe native and recombinant toxins.

Conclusions: The mutant genes were significantly lesseffective in the release of glutamate from rat corticalsynaptosomes than the native and recombinant toxins.

Keywords: Phoneutria nigriventer venom, PnTx2-6 toxin, Site-directedmutagenesis ,10.1016/j.toxicon.2012.04.271

271. Differential Expression of Aquaporin 4 in theHippocampus of Neonate and Adult Rats afterEnvenoming by Phoneutria nigriventer (Ctenidae,Araneomorphae)

Leila Miguel Stavale 1, Edilene S. Soares 1,Monique C.P. Mendonça 1,2, Maria Alice da Cruz Hofling 1,2

1Department of Histology and Embryology, Institute of Biology, StateUniversity of Campinas (Unicamp), Campinas, SP, Brazil2Department of Pharmacology, College of Medical Science, State Universityof Campinas (Unicamp), Campinas, SP, BrazilE-mail address: [email protected] (M. Alice da Cruz Hofling).

Background: Phoneutria nigriventer, popularly knownas armed spider, is responsible for most of the cases ofaraneism in Brazil. Intravenous injection of PNV in ratsresulted in the loss of blood-brain barrier (BBB) perme-ability by impairing transcellular transport and breakingdown the paracellular fence barrier of brain microvesselsendothelium. As result, vasogenic edema by swelling ofastrocytic perivascular endfeet occurs. The swellingresulting from the accumulation of fluid within the brain ismain cause of increased intracranial pressure and subse-quent traumatic brain injury. AQP4, the predominant brain





water channel, is expressed in astrocyte endfeet facingbrain capillaries, perisynaptic spaces and nodes of Ranvier.It is implicated in brain edema formation and resolutionbecause regulates the rapid transport of water within distalfeet of the of perivascular astrocytes, having though a key-role in the maintenance of cerebral homeostasy and waterflow. The aim of this study is to analyze the expression ofAQP4 in the hippocampus, one major target of PNV.

Methods: The time-course analysis (at 2, 5 and 24 hafter PNV intra-peritoneal injection, 1.7mg/kg, n¼6/time)of the immunohistochemical expression of AQP4 inhippocampal regions (CA1, CA2, CA3) of postnatal day 14(P14) and 8-10-week (adult) old rats.

Results: Regional analysis of the immunoreactivity wasdone by GIMP methodology that converts the digitizedimages to grayscale images after color segmentation allow-ing determination of pixels density percentage of the anti-AQP4 immunoreactivity. AQP4 was ubiquitously expressedaround blood vessel walls; also around neuronal bodiesmainly in CA2 and in cell (astrocytes) processes of CA3.

Discussion: The findings revealed that: (i) PNV inducedincrease of AQP4 expression, as compared with controlbaseline, in all three hippocampal regions examined, butthere were regional differences; (ii) The upregulation ofAQP4was higher in CA3/CA2 than in CA1; (iii) Upregulationof AQP4 was more prominent in adult than in P14 neonaterats; (iv) The peak of AQP4 expression occurred chiefly 24 hafter PNV exposure in animals of both ages (with exceptionof CA3 where AQP4 peaked at 5 h).

Conclusions: The water channel protein aquaporin-4plays important role in perivascular astrocyte at the blood-brain interface, likely involved in edema resolution; beingso a molecule targeted by PNV. The time-course anddifferential expression distribution of AQP4 in CA1, CA2 andCA3 suggests spatiotemporal dynamics in the waterhandling in hippocampus after PNV neurotoxic effect andbetween P14 and adult rats.

Financial support: CAPES, CNPq, FAPESP.

Keywords: Edema, hippocampus region, spider venom10.1016/j.toxicon.2012.04.272

272. Cellular and Molecular Demonstration thatVascular Endothelial Growth Factor (VEGF) and itsReceptor Flt-1 and Flk-1 are Involved in Phoneutrianigriventer Envenoming

Monique C.P. Mendonça 1,2, Edilene S. Soares 2,Leila M. Stávale 2, Catarina Râposo 2,Maria Alice da Cruz-Höfling 2

1Department of Pharmacology, College of Medical Science, State Universityof Campinas - Unicamp, Campinas, SP, Brazil.2Department of Histology and Embriology, Institute of Biology, StateUniversity of Campinas - Unicamp, Campinas, SP, BrazilE-mail address: [email protected] (M. Alice da Cruz-Höfling).

Background: VEGF is a major regulator of develop-mental angiogenesis, vascular permeability, and recentlywas also recognized as neurotrophic, neurogenic andneuroprotector, hence being upregulated in many neuro-pathological processes. Evidences that VEGF mediateneurotoxic effect of P. nigriventer venom (PNV) in rats was

lately done by demonstrating the upregulation of Flt-1 inthe CA1, CA2, CA3 and DG regions of hippocampus.

Methods: We investigate the time-course changes (at 2,5, 24 h following i.p. injection of PNV,1.7mg/kg, n¼6/group)of the total amount of VEGF and its receptors Flt-1 and Flk-1proteins by western blotting, as well as their regionaldistribution by immunohistochemistry (IHC) in the hippo-campus of rats (post-natal day 14 (P14) and 8-10 weeks old(adult)). Paired controls were injected with 0.9% saline.

Results: Densitometric analysis of the immunoblots ofthe proteins in the whole hippocampus provided evidencethat VEGF and Flk-1 expression of saline-injected rats isquite the same in P14 animals and that PNV produced nodifference relative to baseline values. PNV also did not alterthe expression of Flt-1 protein in the hippocampus of P14rats. However, PNV promoted gradual increase of Flt-1 from2 to 24 h, which reached statistical significance at 24 h(p<0.05). In 8-10 weeks rats, VEGF and Flk-1 expression alsoshowed a trend for increasing in all periods, which wassignificant just for Flk-1 at 5 h (p<0.05). IHC for anti-VEGFshowed intense labeling of pyramidal cell bodies anddendrites of CA1, CA2 and CA3 hippocampal regions; anti-Flk-1 labeling mirrowed anti-VEGF labeling with differencethat pyramidal cell nuclei are unevenly labeled; anti-Flt-1reactivity was strongly in pyramidal cell nuclei. Vasogenicedema of venules and capillaries were observed in PNV-treated animals whereas it is not seen in saline-treated ones.

Discussion: Studies describing that VEGF affectsepileptiform activities through its receptor Flk-1 and thatFlk-1 up-regulation in pyramidal neurons protects thesecells from hyperexcitability by inhibiting glutamate releaseis in conformity with the present finding.

Conclusion: Since PNV produces convulsion in humansand experimental animals by affecting permeability at BBB,the upregulation of Flk-1 at 5 h in the hippocampus of adultanimals may be an indication that VEGF augment can havea protective role in PNV envenoming through Flk-1 medi-ation. The fact that Flt-1 was upregulated in P14 ratsexposed to PNV is in line with its known higher expressionin neonate rats than in adult rats.

Financial support: CAPES/CNPq/FAPESP/FAEPEX

Keywords: VEGF, spider venom, hippocampus10.1016/j.toxicon.2012.04.273

273. Heterologous Expression of PaluIT1, A CysteineKnot Spider Toxin, in E. coli.

Richa Mehta 1, Kenya Hernández-Salgado 2, Ernesto Ortiz 2,Gerardo Corzo 2, Elba Villegas 1

1UAEM Centro de Investigación en Biotecnología Dept. de ProductosNaturales , Cuernavaca, Morelos, Mexico2UNAM Instituto de Biotecnología, Cuernavaca, Morelos, MexicoE-mail address: [email protected] (E. Villegas).

Background: Low molecular weight spider neurotoxinshave been highly efficient to paralyze or kill insects. Manyof the bioactive components specifically act on targetreceptors in the nervous system of the recipient, such asvoltage gate ion channel, among them the Naþ ion channelis of particular interest for structure and function studies ininsects. PaluIT1 is a 37 amino acid long and four disulfides




bridges peptidewith an ICK structural motif that consists ofa cysteine knot with a triple-stranded beta-sheet. SincePaluIT1 do not recognized mammalian's receptors, it couldbe employed in binding studies to characterize insectreceptors. In addition by labeling the toxin together withspecific directed antibodies, it may be employed to clarifyrelationships between such a ligand and the insect receptorsite. Insect-selective neurotoxins such PaluIT1 are notharmful to humans and have the potential to be used todevelop bio-insecticides; however, they have to beproduced in sufficient amounts first. There are severalmethods to produce them; however, heterologous expres-sion in bacterial cells would be of increased production butit could be a challenge because of the amount of disulfidebridges to be correctly folded, even though some toxinshave been expressed in baculovirus, yeast and bacteria.

Methods: To get sufficient amount of a PaluIT1, a cDNAwasconstructedandcloned into theexpressionvectorpQE30containing a 6His-tag and an FXa proteolytic cleavage region.This recombinant vector was transfected into Escherichia coliBL21 cells and expressed under induction with isopropylthiogalactoside (IPTG). Moreover, a tandem construction ofPaluIT1 (two PaluIT1 cDNA linked with appropriate nucleo-tideswere inserted for R, 5 G and a S residue to cut expressedtoxins), similar plasmids were designed to improve peptideexpression and six different primers were constructed toperform tandem construction by PCR.

Results: Several problems associated with the heterol-ogously expressed toxins containing four disulfide bridgesare discussed. Escherichia coli BL21 frequently mutes theTATAAT sequence inhibiting toxins production, plasmidconstruction must be sequenced in both directions tocorrect this problem. The His-tagged recombinant toxinwas found exclusively in inclusion bodies. Reduced yieldswere obtained after oxidation reaction to fold the toxins. Toavoid inspecific hybridization of cDNA on tandemconstruction short specific primers, are required.

AcknowledgementsThis work was supported by a grant from CONACyT

CB-2008-01 No. 106949

Keywords: Heterologous expression, insecticidal spider toxins10.1016/j.toxicon.2012.04.274

P. Systems & Training

274. A National Serum Depot in the Netherlands;Encountered Antivenom Purchase Difficulties

Kees C.W. van der Zwan 1, Marieke A. Dijkman 2,Ine de Vries 2, Truus W. de Graaf 11 Institute for Public Health and the Environment (RIVM), Bilthoven,The Netherlands2National Poisons Information Center, University Medical Center, Utrecht,The NetherlandsE-mail address: [email protected] (K.C.W. van der Zwan).

Background: Since 2008, a National Serum Depot (NSD)is operational in the Netherlands, guaranteeing antivenomsupply during medical emergencies. The NSD is organizedby the National Institute for Public Health and the

Environment (RIVM), in cooperation with the DutchNational Poisons Information Center (NPIC). The NPICmakesrecommendations concerning the content of the NationalSerum Depot and advices physicians on medical treatmentincluding use of antivenoms. The RIVM is responsible forpurchase, storage and delivery of the antivenoms. Duringestablishment and maintenance of the NSD several anti-venom purchase difficulties are encountered.

Materials and Methods: Like in many countries, anti-venoms are not registered in the Netherlands. There isa permission given by the Netherlands Health Care Inspec-torate (IGZ) to the RIVM to purchase, import, store anddistribute the antivenoms. Because antivenoms are phar-maceutical products, these activities are performed accord-ing to the guidelines of Good Distribution Practice (GDP).

Results: It is a time consuming process to locate andestablish a good relationship with the antivenomproducers. Some antivenom producers do not respondupon (initial) contact. Others have an international saleembargo because of their code of ethics. A main problem isthe availability of antivenoms. Regularly, there are tempo-rarily market failures or the products have only a very shortshelf life left from the moment of purchase. Some productsarrived in bad shape and did therefore not comply with theguideline of GDP leading to the destruction of the product.The package and even the information leaflet of someantivenoms are only available in the local language likeThai, Chinese or Spanish and not in English. In extraordi-nary cases the information on the leaflet was incorrect.

Discussion and Conclusions: In order to have sufficientsupply during medical emergencies, the establishment ofgood working relationships with the producers must betaken seriously. To improve the antivenom market, anti-venom producing companies must apply to the WHOGuidelines for Production Control and Regulation of SnakeAntivenom Immunoglobulins and the general Guideline ofGood Manufactory Practice and Good Distribution Practice.Sharing information between various antivenom depots isimportant to improve the purchase process.

Keywords: Antivenom supply, antivenom market, GxP (GMP and GDP),antivenom acquisition10.1016/j.toxicon.2012.04.275

275. Safe Utilization of Ketamine as a First LineInduction Agent for Rapid Sequence Intubation (RSI) inthe Aeromedical Setting

Janak K. Acharya MD, Cameron L.R. Jones MD, Rais VohraMD, Greg HendeyUCSF-Fresno Emergency Medicine Residency Program, Fresno, CA, USAE-mail address: [email protected] (J.K. Acharya).

Background: Ketamine is a derivative of PCP that acts asa dissociative anesthetic. It has a number of benefits as ananesthetic and in terms of its effects on the physiology ofcritically injured patients in the prehospital settingrequiring RSI.

Objectives: To show that ketamine can safely be used asa first line induction agent for RSI in hypotensive tonormotensive patients requiring aeromedical transport.



Fig. 1.

Fig. 2.


Methods: Retrospective case series over 6 month periodfollowing the introduction of ketamine into aeromedicalprotocols.

Results: 7 of 18 patients in the case series receivedketamine as an induction agent. 100% percent of patientsreceiving Ketamine avoided hypotensive sequelae. 0%developed hypertension or other adverse effects. Vitals andend tidal CO2 improved in all patients receiving ketamine.

Conclusions: Ketamine was a safe and beneficial alter-native to existing induction agents in hypotensive traumapatients being transported by air.

Keywords: Ketamine, aero-medical, RSI10.1016/j.toxicon.2012.04.276

276. Circus Venomous: An Interactive Tool forToxinology Education

Rais Vohra 1,2, Susanne Spano 1

1UCSF-Fresno Medical Center, Fresno, CA, USA2California Poison Control System, Fresno-Madera Division, Fresno, CA, USAE-mail address: [email protected] (R. Vohra).

Background: Clinical education about envenomationsand their treatment may convey clinical and zoologicaldetails inadequately or flatly. In recent years, the wide-spread availability of models and videos of venomousspecies have created unique opportunities for toxinologyeducation.We share our experiences using a new toolkit foreducating a diverse array of clinicians, students, andwilderness medicine enthusiasts.

Methods:We examined the cost, number of participants,and satisfaction data since the initiation of a portable work-shop featuring high-fidelity exhibits of venomous species.Termed the “Circus Venomous,” this educational toolkitconsists of several boxes of props, such as plastic models,photos, and preserved specimens of injurious species (seeTable).

The workshop consists of three phases: 1.) participantsview all exhibits and answer clinical questions regardingvenomous injuries; 2.) short video clips from television andcinema are viewed together, andmyths about envenomationinjuries are debunked; 3.) debriefing session and wrap-up.

Results: We have utilized the Circus Venomous to teachmedical students, residents, practicing community clinicians,nurses, PAs, national and regional parkmedics, and wilder-ness enthusiasts. The major cost (about $800) was spent onthe purchase of highly durable, lifelike models and well-preserved real reptile and arachnid specimens.When formalfeedbackwas solicited, theparticipants expressed high levelsof satisfaction, scoring an average of 4.3, 4.4, and 4.3 out of 5points in the respective areas of content, presentation, andpractical value of the activity. Sincewe have used this exhibitwith approximately 250 participants over 2 years, we esti-mate the materials cost per participant is approximately $3.

Conclusions: The Circus Venomous is a novel, interactive,flexible, and cost-effective teaching tool about envenomationemergencies.We hope that this conceptwill encourage otherclinical educators towards further innovation. Future direc-tions forourgroup includegreater inclusionofmarine speciesinto the Circus Venomous, and formal longitudinal testing tomeasure knowledge retention based on this approach.



Fig. 3.

Table: Inventory of Circus Venomous, An Interactive Tool for Toxinol-ogy Education

western diamondback rattlesnakedpreserved specimencoral snakedplastic modelnon-venomous speciesd2 plastic model and 1 preserved specimencoral snake, heloderm lizard, and caterpillarsdhigh-quality imagesgrizzly and black bear skull – plastic replicaspreserved arachnidsdmounted specimenscommercial snakebite “first aid and extraction kit”splinting and wrapping materialsprotective and inadequate footwear for wilderness expeditions

Keywords: Toxicology, instructional tools, envenomation education10.1016/j.toxicon.2012.04.277

277. Prehospital Management of Envenomations in theState of Queensland, Australia

John L. Rathbone 1,2, Jamie Quinn 3

1Queensland Ambulance Service, Townsville, Australia2Marine Stinger Advisory Panel, Queensland, Australia3Australian Centre for Prehospital Research, Queensland Ambulance Service,Brisbane, AustraliaE-mail address: [email protected] (J.L. Rathbone).

Background: Queensland is the second largest state inAustralia and covers an area of 1,730,648 km2. Because ofits vast size, there is significant variation in climate acrossthe state and a range of landscapes from coastal environ-ments, deserts, bush, rainforest, mountainous regions andlarge urban centres. Queensland is home to a variety ofspecies of venomous wildlife. The Queensland AmbulanceService (QAS) provides emergency treatment to patientswho experience bites, stings and evenomations. Para-medics are dispatched in road ambulances, aircraft (heli-copters and fixed wing) and marine craft to provide care topatients in a range of settings.

Method: This study investigates and profiles bites,stings and envenomations treated by QAS paramedics 20072011. Retrospective analysis of QAS patient data wasundertaken. A manual audit of box jellyfish stings and

Irukandji stings was performed to evaluate the effective-ness of paramedic treatment with antivenom and magne-sium sulphate.

Results: The QAS attended 8,741 cases of bites, stingsand envenomations 2007-2011. The majority of seriousinjuries were dog bites, snake bites, spider bites, and fish orjellyfish. Morphine was administered to 582 patients forsnake bite (n¼117); spider bite (n¼77); dog bite (n¼61);jellyfish or fish sting (n¼55) and other injury types. Ofpatients who received pain relief (n¼1,214 methoxyfluraneand /or morphine), a mean reduction in pain of 3.03 points(using the 10 point scale) was observed. Regarding seriousmarine envenomations, the QAS treated 51 patients withmagnesium sulphate for Irukandji stings and achieveda mean pain reduction of 4.66 points in these patients.Eight patients were treated with box jellyfish antivenom,with a mean pain reduction of 2.2 points. An audit of theoutcomes for these patients will be presented, with anexamination of the efficacy of magnesium sulphate and boxjellyfish antivenom in these patients who have suffereda serious and painful injury.

Discussion: The preliminary results show a significantdiversity in envenomation types across a range of terrainsin Queensland. Given the diversity of environments andenvenomation types, paramedic education and training isregionally focused to ensure that paramedics can deliverthe most appropriate front-line treatment for patients.

Conclusion: Because the geographic area of Queenslandcovers a range of environments the delivery of appropriateand standardised ambulance services poses significantchallenges. The results of this analysis demonstrate that theQAS, in line with a commitment to evidence-based prac-tice, has reflected the treatment protocols in hospitalsettings and successfully equipped paramedics tocommence definitive treatment for envenomed patientsbefore transporting to hospital.

Keywords: Ambulance, treatment, envenomations10.1016/j.toxicon.2012.04.278

278. Epidemiologic Situation of Envenomation byVenomous Animals in Argentina. 2007-2011 Period

Natalia Casas 1, Laura Geffner 1, Horacio Echenique 1,Vanessa Costa de Oliveira 1,2, R. de Adolfo Roodt 2,31Dirección de Epidemiología, Ministerio de Salud de la Nación, Argentina2 Laboratorio de Toxinopatología, Centro de Patología Experimental yAplicada, Facultad de Medicina, U.B.A, Argentina3 Instituto Nacional de Producción de Biológicos, Administración Nacional deLaboratorios e institutos de Salud, ArgentinaE-mail address: [email protected] (R. de Adolfo Roodt).

Background: Intoxications from venom inoculation byvenomous animals are of mandatory notification inArgentina. Major incidence of injuries of toxicologicalsignificance, are caused by species of the genera Tityus(scorpion), Lactrodectus, Loxosceles and Phoneutria (spiders)and Bothrops (Rhinocerophis, Bothropoides and Bothrops),Crotalus and Micrurus (snakes). The aim of this work is todescribe the national situation of envenomation byvenomous animals, contributing to the development of the




surveillance network and consolidation of the informationavailable about mortality and morbidity.

Methods: Study design was descriptive and transversal,and data from the period 2007-2011 was collected from theNational Surveillance System of Health (Sistema Nacionalde Vigilancia de la Salud; SNVS). Analysis was made usingMicrosoft Office Excel, GeCo C2, Epiinfo 3.5.1 and SIGEpi 1.0software.

Results: Within the study period 45012 cases of enve-nomation by venomous animals (9002 cases/year) wereregistered in the SNVS. 3692 (8.2%) cases were caused bysnakes, 6084 (13.5%) by spiders and 35236 (78.3%) byscorpions, corresponding to an incidence rate of 1.8, 3.0 and17.6 cases/10.000 inhabitants per year, respectively. 15-24years old was the most affected age group. During the sameperiod 40 deaths were registered, snake bites accounted for42.5%, scorpion stings for 35.0% and 22.5% were caused byspider bites. Among snake caused deaths, people older than65 years old were most affected (47.1%), while spiderscaused death preferably to the patients older than 45 yearsold (62.5%). On the other hand, children of 9 years old orless accounted for the majority of deaths by scorpions(96.4%). Geographic distribution of envenomation inci-dence is not uniform, while scorpions and spiders show thehighest incidence in the northwest region (58.5 cases/10.000 inhabitants per year and 7.7 cases/10.000 inhabi-tants per year respectively), snakes morbidity is higher inthe northeast region (8.6 cases/10.000 inhabitants peryear). Besides, Northeast and Northwest regions show thegreatest notification for snakes (1591 and 1379 casesrespectively), while scorpion and spider notification ishighest in the center and northwest regions (14033 and13757 cases for scorpion; 1718 and 1799 for spiders).Envenomation occurs mainly in the summer season for allthree poisonous animals.

Conclusions: Envenomation by venomous animalsconstitutes a significant health problem in some regions ofArgentina. These events are always medical emergenciesand prevention is made through education. Besides,specific treatment provision and adequate management ofpatients are necessary to avoid serious damage or death.

Keywords: Health surveillance, snakes, spiders, scorpions, epidemiology10.1016/j.toxicon.2012.04.279

279. Travel Toxinology: An Illustrative Case of BrownSpotted Pit Viper (Protobothrops mucrosquamatus) BiteWith Review of Clinical Toxinology Issues in TravelMedicine

Julian White 1, Bart Currie 2

1 Toxinology Dept., Women's & Children's Hospital, North Adelaide,SA, Australia2Menzies School of Health Research, Casuarina, NT, AustraliaE-mail address: [email protected] (J. White).

Background: Tourism is emerging as a major economicdriver globally and travellers visit ever more exotic places.As a consequence, exposure to Venom Induced Diseases(VIDs) becomes an increasing risk. Lack of resources to

adequately diagnose and treat VIDs in many parts of theworld increases the risk of suboptimal outcomes.

Case Report: A 12 year old girl on holiday with herfamily in Vietnamwas bitten on her ankle by a snake whileon a tour boat in Ha Long Bay. Local suction and a tourni-quet were applied as first aid, then she was transferred tothe mainland hospital where the tourniquet was removed,but no antivenom given as the snake identity wasunknown. The snake had been killed and was brought withthe patient. The patient complained of significant local painwhich remained untreated, thenwas transferred to a largerhospital, where again no antivenom was given because ofdoubt over the snake‘s identity. By this time, many hourslater, the foot and leg, extending to the abdomen, wasmarkedly swollen. A mild coagulopathy was present, withthrombocytopenia and anaemia. The patient continued tobe managed conservatively, with no antivenom and aftera week was transferred to Australia. There a marked footdrop was evident with MRI evidence of deep foot musclenecrosis. Despite this over the following weeks the patientregained full use of the foot. Clear photographs of the killedsnake were identified by experts as Protobothrops mucros-quamatus. The only antivenom for this snake, a knowninhabitant of northern Vietnam, is made in Taiwan.

Discussion: This case illustrates a number of problemsfor tourists envenomed in another country wherecommunication and health system issues can adverselyaffect treatment. Early consultation with a clinical tox-inologist might have allowed identification of the snakeand sourcing of suitable antivenom. This case is illustrativeof a wider problem for “travel toxinology” cases. Localhealth systems may not have the expected expertise todiagnose and treat envenoming, even by local species.Rapid access to (clinical toxinology) expertise per phone,availability of suitable antivenoms and urgent transfer ofstabilised envenomed patients are all required elements inimproving patient outcomes.

Keywords: Snakebite, Protobothrops mucrosquamatus, envenoming10.1016/j.toxicon.2012.04.280

Q. Venomous Animal Biology

280. Genetic Regulation of Venom Production duringEmbryonic Development of the Indochinese SpittingCobra, Naja siamensis

Jessica M. Logan 1, Peter J. Mirtschin 1,2, Anthony E. Woods 1

1 School of Pharmacy and Medical Sciences, University of South Australia,Adelaide, SA, Australia2Venom Supplies Pty Ltd, Tanunda, SA, AustraliaE-mail address: [email protected] (J.M. Logan).

Background: Snake venoms are a potential source ofpharmacologically active components. Since the initialdiscovery of the ACE inhibitors which were originallyderived from the venom of the Brazilian Arrow Head Viper,several compounds have stimulated pharmacologicalinterest although issues relating to venom yield and vari-ability have hampered efforts to isolate these agents.




Ideally it would be beneficial to produce the venom in an invitro model which would allow the desired components tobe engineered in suitable amounts. However, to date,fundamental knowledge of the mechanisms underlyingvenom production and its regulation is not available.

Methods: Naja siamensis clutches (n¼4) were obtainedand embryos removed at varying stages of development.Venom proteins were demonstrated (using therapeuticantivenom as an antibody source) via a direct immuno-histochemical technique. Embryos were also screened forgene expression using in situ hybridisation with DIGlabelled RNA probes targeting sequences responsible forrepresentative proteins within the venom.

Results: Production of venom components was identi-fied in 3 of 4 embryo clutches. Histological examinationrevealed differences in gland morphology and overalldevelopment (between embryos from separate clutches)despite identical post-oviposition times. Gene expressionwas demonstrated in all Naja siamensis clutches at stageswhen the venom glands displayed similar histologicaldevelopment. A 12 day delay was observed between theinitiation of gene expression to the demonstration ofvenom proteins.

Discussion: The initiation of gene expression wasdetected at different post-oviposition time points acrossthe embryo clutches. This was likely due to variabledevelopment stages within the clutches as major pheno-typic differences were observed histologically at the sametime post-oviposition preventing correlation betweenclutches. The 12 day lag between initial gene expressionand identification of the venom protein product suggestseither post-transcriptional processing or post-translationalmodification is occurring.

Conclusions: The efforts to elucidate the primary stagesof venom production were restricted by the lack ofphenotypic correlation between the embryo clutches at thesame time points post-oviposition. The need for a correla-tion strategy is clearly evident however the initial timing ofgene expression leading to protein production has beensuccessfully demonstrated. This has lead to several infer-ences about its importance in venom production, compo-sition and yield.

Keywords: Venom, genes, embryo10.1016/j.toxicon.2012.04.281

281. The Desert Massasauga (Sistrurus catenatusedwardsii): Biology and Venom Biochemistry

Stephen P. MackessySchool of Biological Sciences, University of Northern Colorado, Greeley,CO, USAE-mail address: [email protected].

Background: The Massasauga (Sistrurus catenatus) isa small rattlesnake which occurs in grasslands of NorthAmerica, from northern Mexico to southeastern Canada.Although threatened in many parts of its range, the dimin-utiveDesertMassasauga (S. c. edwardsii) remains abundantatseveral locations in more mesic regions of the shortgrasssteppe of southeastern Colorado. Our numerous studies of

the ecology/natural history and venom biochemistry/geno-mics make Desert Massasaugas one of the better-character-ized species of rattlesnakes, and this summary will examinethe interplay of animal biology and venom biochemistry.

Methods: Snakes (750) were collected on a privateranch in southeastern Colorado and were processed in thelab (morphometrics, venom extraction and PIT-tagging; 12snakes were also implanted with radios for telemetrystudies). Massasaugas were radiotracked for two yearsduring the active season to determine spatial ecology andhabitat use. Venoms were subjected to a variety ofbiochemical and proteomic analyses, and two snakes weresacrificed for transcriptomics studies.

Results: Massasaugas make strongly directional migra-torymovements which are resource-driven. Abundant prey(lizards, centipedes, rodents) and favorable thermoregula-tory sites occur in the summer habitat, while stablehibernacula exist in the shortgrass habitat. Long-termmark/recapture studies indicate that Desert Massasaugasare abundant but short-lived, with average adult survi-vorship of <4 years. Desert Massasauga venom is quitepotent toward both lizards and rodents, and it is muchmore toxic to a common rodent prey species (Perognathus)than toward rodent species not utilized (Peromyscus). Pro-teomic and genomic analyses indicate that a crotoxinhomolog, characteristic of other type II rattlesnake venoms,is not expressed in this species, but genes for 5 isoforms ofthree-finger toxins (3FTXs) are present. Unlike most vipervenoms, only one dominant isoform of an acidic PLA2 ispresent in venom of S. c. edwardsii.

Discussion: The Desert Massasauga is one of only a fewspecies of viperids demonstrated to possess genes for 3FTXs,a protein family which includes the potent a-neurotoxins ofelapids and several taxon-specific neurotoxins of some rear-fanged snakes. However, 3FTxsdonot appear to beexpressedin the venom. Based on 2D SDS-PAGE, over 100 proteinscomprise this venom, and serine proteinases (thrombin-like,kallikrein-like) are abundant components which maycontribute to high venom lethality in mice and lizards.

Conclusion: The Desert Massasauga in Colorado isan excellent model species for evaluating influences ofnumerous ecological factors on venom evolution, andcontinuing studies are investigating population levels ofvenom and genetic variation.

Keywords: Ecology, toxin evolution, proteome10.1016/j.toxicon.2012.04.282

282. Sexual Variation in Timing of Egress and Ingress inTiger Rattlesnakes (Crotalus tigris)

Chip Cochran 1, Matt Goode 2

1Department of Earth and Biological Sciences, Loma Linda University, LomaLinda, CA USA2 School of Natural Resources, University of Arizona, Tucson, AZ, USAE-mail address: [email protected] (C. Cochran).

Background: Temperate zone snake species avoidhazardous winter temperatures by overwintering in ther-mally sheltered hibernacula. The tiger rattlesnake, Crotalustigris, is a summer mating, medium-sized pitviper that




ranges from southern Sonora northward into south-centralArizona.

Methods: With the aid of temperature-sensitive radiotransmitters we tracked tiger rattlesnakes in the Rincon(two sites) and Tortolita Mountains (one site) of PimaCounty, Arizona from 1997-2010.

Results: Based on data pooled across years and sites, wedetermined that timing of ingress coincides for males andfemales. However our results revealed that timing of egressvaried, with females leaving dens significantly earlier thanmales. Specifically, the average date of emergence forfemales was almost a full month (22-23 March) beforemales (20-21 April). Due to small sample sizes from ourRincon Mountain sites, we were only able to compareinterannual variation in the timing of egress at our TortolitaMountain site from 2004-2006. In each year, femalesemerged (3/19/2004, 3/16/2005, 3/28/2006) earlier thanmales (4/12/2004, 3/31/2005, 5/1/2006).

Discussion: Our data provides support for the hypoth-esis that male rattlesnake species which do not mate inspring should not emerge before females. Unlike females,there is no need to come out to bask in early spring tofacilitate the process of gametogenesis. We are also inves-tigating the possibility of sexual variation in overwinterbody temperature in C. tigris to further our understandingof this species winter ecology.

Keywords: Rattlesnake, ecology, ingress, egress10.1016/j.toxicon.2012.04.283

283. Relocator Proteins: Identification of the ChemicalComponent of Venoms Allowing Prey Recovery DuringStrike-induced Chemosensory Searching

Anthony J. Saviola 1, Stephen P. Mackessy 1, David Chiszar 2,Chardell Busch 2

1 School of Biological Sciences, University of Northern Colorado, Greeley,CO, USA2Department of Psychology, University of Colorado, Boulder, CO, USAE-mail address: [email protected] (A.J. Saviola).

Background: Among advanced snakes, a chemicalmode of dispatching prey is commonly utilized to obtainprey rapidly and with minimal contact. Venoms containa variety of protein, peptide and small organic compounds,and a persistent issue in the study of venom evolution hasbeen to explain the compositional complexity of venoms.Lethal toxicity toward particular prey has demonstratedthe adaptive significance of several taxon-specific venomcomponents, but for most venom proteins, particularly thelow toxicity, non-enzymatic fractions, a well-defined rolein envenomation and predation has not been established.During predatory episodes, rattlesnakes and other pit-vipers commonly strike, envenomate and release prey,minimizing retaliation. They are then confronted with theadditional task of relocating the carcass via chemosensorysearching once venom has taken its course, which they dowith a high degree of precision. However, the specificcomponent of venom involved in altering the chemical cuesof prey, allowing for relocation, has yet to be identified.

Methods: Two-hundred and fifty milligrams of crudeCrotalus atrox (Western Diamondback) venom was frac-tionated using a BioGel P-100 size exclusion column. Allfractions were tested for enzymatic activity of enzymescommon to rattlesnake venoms. Peaks were dialyzed,lyophilized, and frozen at -20 C until behavioral testing.Eight C. atrox were tested for vomeronasal chemosensoryresponses to fractionated peaks. The significant peak (peakIII) was further fractionated using RP-HPLC, and proteinidentification was confirmed by MALDI-TOF mass spec-trometry and N-terminal sequencing.

Results: Enzymatic and other major protein compo-nents of size exclusion-fractionated C. atrox venom had noeffect on discrimination of envenomated vs. non-enveno-mated prey by snakes. Conversely, peak III elicited a statis-tically significant response to treated prey. Furtherpurification by RP-HPLC and analysis by MALDI-TOF massspectrometry and N-terminal sequencing confirmed thatthis peak contained only two monomeric disintegrins,crotatroxins 1 and 2.

Discussion: In the field, this chemical tag on prey willhelp minimize foraging time and greatly expeditediscrimination of a trail left by envenomated prey from themany trails of non-envenomated conspecifics. Our resultsdemonstrate unequivocally that venom components, suchas disintegrins, can have important biological roles whichextend beyond those that are apparent from their bio-chemically functional roles.

Conclusion: This is the first study to identify thecomponent of venom allowing for relocation of enveno-mated prey, and we suggest that a major biological role ofvenom disintegrins for rattlesnakes is to allow these strike-and-release predators to relocate envenomated preyeffectively.

Keywords: Chemosensory searching, disintegrins, prey relocation10.1016/j.toxicon.2012.04.284

284. The Bio-Logic of Venom Complexity

David Morgenstern 1,4, Brett Hamilton 2, Daniel Sher 3,4,Alun Jones 1, Gideon Mattius 4, Eli Zlotkin 4, Deon Venter 2,Glenn F. King 1

1 Institute for Molecular Bioscience, University of Queensland, St. Lucia 4072,Australia2Department of Pathology, Mater Health Services, Raymond Terrace, SouthBrisbane, 4101, Australia3Department of Marine Biology, School of Marine Sciences, University ofHaifa, Haifa, Israel4Department of Cellular and Animal Biology, Silberman Life Science Institute,Hebrew University, Jerusalem, IsraelE-mail address: [email protected] (D. Morgenstern).

Background: The dependency of venomous species ontheir venom for prey capture has resulted in the evolutionof very complex mixtures. Although this strategy is effec-tive, the use of venom has an associated metabolic pricetag, and consequently venomous animals tend to use theirprotein-rich venom sparingly. While complexity appears tostand in contrast to the need for metabolic economy, westill lack knowledge as to the significance of this complexity




for venom use. The aim of the present study was to char-acterize the molecular complexity of natural envenoma-tions and to examine the contribution of individual venomcomponents to overall venom toxicity.

Methods: We used the Australian funnel-web spiderHadnyche infensa and the scorpion Hottentotta judaicus asmodels of two independently recruited venom systems inorder to characterize venom use under natural conditions.In both cases, the animals were provoked to repetitivelydeliver venom, simulating a defensive secretion, leading tothe gradual release of multiple venom droplets. The indi-vidual venom droplets were then assayed for their toxicityagainst flesh fly larvae (Chrysomya megacephala). Therelative abundance of the various components betweensecretions of each series was assayed using LC-ESI-MS.Additionally, we have examined the distribution of thevarious components identified previously in the venom, inthe venom gland using MALDI imaging.

Results and Discussion: an analysis of the individualsecretions, found them to be extremely heterogeneouswith regard to their volume, protein content, proteinprofile, and toxicity. Nevertheless, a pattern was observedwhereby the toxicity of the stings peaks with time. Thisincrease correlated with a discernable change of venomcomposition, whereby the most potent toxins are secretedonly in later stings. The correlation between toxicity andpeptide abundance reveals that only a fraction of the toxinscontribute significantly to venom toxicity. Remarkably, wedemonstrate that this manipulation of the venom compo-sition is achieved by a spatial organization of toxinproduction within the venom gland. Imaging mass spec-trometry revealed that the most potent toxins, which areabundantly secreted only late in the secretion series, arestored deeper within the gland than the toxins seen in theinitial venom secretions.

Conclusion: We suggest that this arrangement allowsvenomous arachnids to reduce the selection pressure ontheir most potent toxins through a reduction in the occa-sion of their use. This strategy appears to be successfulenough to have evolved at least twice, though preliminaryevidence exists to suggest this modulation may also occurin spitting cobras, implying an evolutionary favorablestrategy

Keywords: Venom complexity, venom optimization, proteomics10.1016/j.toxicon.2012.04.285

R. Venomous Animal Collections

285. Venomous Workshop: Evolution of a ProfessionalTraining Course

Douglas L. HotleAlbuquerque BioPark, Department of Herpetology, Albuquerque, NM, USAE-mail address: [email protected].

Background: Venomous animals pose a myriad ofunique challenges for those who work with them. Frompropagation to politics, few other animals are surroundedwith such mystique and misinformation.

Methods: The Albuquerque BioPark in conjunctionwithspecialists from around the world have funneled theirexpertise into this week long workshop. Covering topicsincluding handling, exhibit design, fieldwork, envenoma-tions, antivenom, venomics and more, this workshop isa first of its kind. All types of venomous animals will becovered including marine life and terrestrial invertebrates.Theworkshop is open to zoo staff, field biologists, academicresearchers, Federal and State authorities and even theserious private enthusiast.

Results: There has been an appreciable amount ofinterest in this workshop with attendees expected fromcountries outside of the United States as well.

Conclusions: A workshop covering all aspects ofvenomous animal handling has drawnwidespread interest.Results will be reported in future presentations.

Keywords: Venomous animals, professional training10.1016/j.toxicon.2012.04.286

286. Legal Conundrums Impeding Patient SafetyInitiative to Prevent Exotic Envenomation in the UnitedStates

Joshua Z. Silverberg 1,2, Michael Touger 1, Donal M. Boyer 31 Jacobi Medical Center, Department of Emergency Medicine, Albert EinsteinCollege of Medicine, Bronx, NY, USA2Montefiore Medical Center, Department of Emergency Medicine, AlbertEinstein College of Medicine, Bronx, NY, USA3Department of Herpetology, Bronx Zoo/Wildlife Conservation Society, Bronx,NY, USAE-mail address: [email protected] (J.Z. Silverberg).

Background: During 2011 In New York state there werefifty-seven venomous snakes confiscated comprising forty-nine species of world-wide origin after the possible suicideof their owner. The owner was found with puncturewounds consistent with a snakebite and her blackmamba's(Dendroaspis polylepis) enclosure was unlocked. The largecollection was brought to the Bronx Zoo and placed inquarantine. This presentation will discuss how currentfederal state and local law affects the availability ofvenomous reptiles and the clinical relevance of thatavailability.

Discussion: There is currently limited federal lawregarding the importation, possession and interstate trans-port of these animals. A great variability between states inthis area exists. An example pertinent to this case is therelatively strict regulations of NewYork State as compared tonearby states–especially Pennsylvania. To possessa venomous reptile in New York State, a permit from theNew York Department of Environmental Conservation isrequired. Outside of a few local municipalities there is noregulation in Pennsylvania. There are large snake expos inWestern Pennsylvania that have been responsible formultiple envenomations in the New York Metropolitan areabased on feedback from snakebite patients. This inconsistentregulatory situation makes primary preventative measuresagainst snake envenomation difficult. Recently, federalregulations banning the interstate transport of largeconstrictor snakes have been adopted. A proposal for similar




federal regulation regarding venomous reptiles is likely to becontroversial.

Keywords: Venomous snakes, envenomation, regulation10.1016/j.toxicon.2012.04.287

287. Logistics and Problems in Managing a LargeConfiscation of Venomous Snakes

Donal M. BoyerDepartment of Herpetology, Bronx Zoo, New York, NY, USAE-mail address: [email protected].

Background: The New York Department of Environ-mental Conservation (DEC) contacted the Bronx Zoo onJune 14th 2011, requesting assistance for the removal andtemporary maintenance of a large confiscation ofvenomous snake held at a private residence. Local police inPutnam County were investigating an apparent suicide anddiscovered a collection of 17 elapids, 10 vipers, 29 pit vipersand one rear fanged colubrid).

Methods: Bronx Zoo Herpetology Department Staffconfirmed identification of species and removed collectionto a secure quarantine facility at the Bronx Zoo. DECprovided chain of custody document granting transferwhile enforcement officials worked to resolve the case.Dr Michael Touger, Jacobi Medical Center's SnakebiteTrauma Unit and Bronx Zoo consultant, was apprised of theconfiscation. He then began identifying appropriate anti-venom and locations for products we might lack. As staffidentified snakes with potential health problems ourveterinary staff beganmedical work up. Routine quarantinediagnostics were started.

Results: During the quarantine period fecal samplesrevealed several endoparasites; Strongyles, rhabditidnematodes, flagellated protozoa and Cryptosporidium,these were treated with appropriate drugs. Snake feceswere screened for the presence of Cryptosporidium throughindirect immunofluorescent antibody staining and whenpositive, repeat fecal samples were sent for cryptosporidialspecies identification, as determined through PCR. Severalspecimenswere found to have serious health problems thatresulted in euthanasia. These snakes underwent completenecropsies. A portion of the live collectionwas screened forophidian paramyxovirus, using PCR on lung wash samples.Because many of these snakes were fastidious eaters andonly defecated occasionally, collection of enough fecalsamples to meet routine quarantine testing requirementswas a long arduous task. When the quarantine period wascomplete we began the process of distributing the snakesto other AZA zoos.

Discussion: When wildlife agencies confiscatevenomous snakes they are often not able to care for thesereptiles and must turn to zoos for assistance. An alternativeoption is euthanasia of the confiscated specimens whichhas occurred in some instances. The commitment of stafftime to care for these additional snakes was significant. Thebenefit was the opportunity it created for staff to gainadditional training and experience with venomous speciesnot in our collection. A few specimens were incorporated in

our collection. The commercial availability of thesevenomous snakes and illegal private ownership resultedtragically in this case.

Keywords: Confiscation, quarantine, zoo10.1016/j.toxicon.2012.04.288

288. Unique Challenges Faced During the Creation ofa New Herpetology Department with New VenomousAnimal Species and Safety Policies within the City ofVirginia Beach's Virginia Aquarium and Marine ScienceCenter

William G. HarshawVirginia Aquarium and Marine Science Center, Department of Mammals andHerpetology, Virginia Beach, Virginia, USAE-mail address: [email protected].

Background: Our 25-year-old, city-owned aquariuminitially housed 1 native venomous snake species “Cane-brake rattlesnake” [Crotalus horridus atricaudatus]. Overtime, we added two additional, native exhibits featuring“Cottonmouths” [Agkistrodon contortrix] and “Copper-heads” [Agkistrodon piscivorus]). We had no dedicated/skilled herpetology staff and only two individuals capableof working directly with venomous vipers. In 2003 webegan planning an expansion that would house TomistomaCrocodiles (Tomstoma Schlegelii), Komodo dragons (Varanuskomodoensis), Egyptian cobras (Naja haje), Fat-tail scor-pions (Adroctonus australis hector), Green Pitvipers (Cryte-lytrops albolabris), a walkthrough Red Sea aquarium tunneland numerous other exhibits.

Challenges and Responses: Adding exotic venomousspecies to our native vipers required the development ofa new institutional herpetology department, with newsystems and protocols. This included the recruitment andtraining of animal care staff, the acquisition of non-nativeantivenoms, as well as issues related to their storage andpotential use, event preparedness protocols, and EMS andhealthcare provider outreach. An initial training sessionwas held with the assistance of the regional poison centerand an EMS consultant from the Miami-Dade AntivenomUnit. Regional physicians, local universities, EMS responseteams, animal control and aquarium officials were invited,with CME made available to participating physicians. Asecond training program was presented to the local EMSproviders and envenomation management protocolsdeveloped and approved for use. Acquiring non-nativeantivenoms can be difficult and labor intensive. Weacknowledged that antivenoms, if available, should beobtained prior to venomous animal acquisition. Particulardifficulties were encountered in acquiring SCORPIFAV,which would provide coverage in the event of a sting fromthe Fat-tail scorpion. Suppliers for this antivenom werelimited and very difficult to contact. Acquisition of thisantivenom required more than two years to accomplish.Despite a hobbyist collection of this species, no U.S. zoosstocked this antivenom and, to our knowledge, the onlyother available antivenom in the U.S. is through Miami-Dade Antivenom Unit.




Conclusions: The development of a venomous animalcollection, particularly one housing non-native venomousspecies, presents challenges in animal selection, antivenomacquisition, staff recruitment, training and safety policies,envenomation management protocols, and the involve-ment of regional medical professionals.

Keywords: Herpetology, protocol development10.1016/j.toxicon.2012.04.289

289. 2011 Putnam County Venomous SnakeConfiscation: Antivenom Coverage Decisions

Michael Touger 1,2, Donal M. Boyer 31 Jacobi Medical Center, Department of Emergency Medicine, Bronx, NY USA2Albert Einstein College of Medicine, Bronx, NY USA3Herpetology Department, Bronx Zoo/Wildlife Conservation Society, USAE-mail address: [email protected] (M. Touger).

Background: In 2011, Putnam County, NY, confiscated57 highly venomous snakes of world-wide origin.

Methods: Descriptive case series.Results: For seven animals, adequate available species-

specific antivenom supply was lacking. Forty-four animalsare to be dispersed to other institutions. Ten animals are tobe kept as part of the permanent collection. Three animalsdied of causes unrelated to the confiscation.

Discussion: Confiscation of highly venomous snakesresults in scenarios that require clinical decision making,guided by availability of exotic antivenoms that arerequired for staff safety.

Conclusion: Overall the antivenom stocks at the BronxZoo provided excellent coverage for the majority of theconfiscated animals.

Keywords: Snake confiscation, antivenom supply10.1016/j.toxicon.2012.04.290

290. Microbiological Evaluation of Different Strategiesfor Management of Snakes in Captivity

M.V. Michelle Vanessa Campagner 1,2, Sandra M.G. Bosco 3,Eduardo Bagagli 3, Maria de Lourdes R.S. Cunha 3,Bruna C. Jeronimo 2, Eduardo Saad 1,2, Natalia P. Biscola 1,2,Rui Seabra Ferreira-Junior 1,2, Benedito Barraviera 1,2

1Department of Tropical Diseases, Botucatu Medical School, São Paulo StateUniversity, (UNESP – Univ Estadual Paulista), São Paulo State, Brazil2 The Center for the Study of Venoms and Venomous Animals (CEVAP), SãoPaulo State University, (UNESP – Univ Estadual Paulista), São Paulo State,Brazil3Department of Microbiology and Immunology, Botucatu BioscienceInstitute, São Paulo State University, (UNESP – Univ Estadual Paulista), SãoPaulo State, BrazilE-mail address: [email protected] (B. Barraviera).

Background: Keeping snakes in captivity to producevenom for scientific research and production of inputs isnow a worldwide practice. Maintaining snakes in captivityinvolves capture, infrastructure investments, managementtechniques and appropriate qualified personnel. Further-more, the success of the project requires knowledge of

habitat, nutrition and reproduction, and control of oppor-tunistic infections.

Methods: This study evaluated the management ofsnakes in three types of captivity (quarantine, intensive andsemi-extensive) and diagnosed bacterial and fungalcontaminants. A bacteriological profile was obtained byswabbing the oral and cloacal cavities, scales and venoms ofhealthy adult snakes from Bothrops jararaca (Bj) and Cro-talus durissus terrificus (Cdt).

Results: There was predominance of Enterobacteriaceae,especially non-fermenting Gram-negative bacilli excludingPseudomonas spp, Gram-positive bacteria and non-ferment-ing Gram-negative bacilli. Statistically, intensive captivityresulted in the highest number of bacterial isolates, followedby recent capture (quarantine) and by semi-extensivecaptivity. No statistical difference was found between Bj andCdtbacterial frequency. Invitrobacterial susceptibility testingfound the highest resistance against the semi-syntheticpenicillins (amoxicillin and ampicillin) and highest sensi-tivity to amicacin and tobramicin amynoglycosides. To eval-uate mycological profile of snakes from intensive captivity,collections were obtained from two healthy Bj and one B.moojeni, one B. pauloensis and one Cdt showing whitishlesions on the scales suggestive of ringworm. Usingconventional methods and DNA-based molecular proce-dures, five samples of Trichosporon asahii were identified.

Conclusions: Despite the traditional role of intensecaptivity in ophidian venom production, semi-extensivecaptivity was more effective in the present study by virtueof presenting superior control of bacterial and fungaltransmission, easier management, cheaper cost anddecreased mortality; therefore, it should be considereda good alternative for tropical countries.

Keywords: Bothrops jararaca, Crotalus durissus terrificus, Trichosporon asa-hii, Enterobacteriaceae, management in captivity10.1016/j.toxicon.2012.04.291

291. The Dilemma: Balancing Antivenom Cost vs.Investment in Conservation

Jessi Krebs, Mary M. CederstrandOmaha's Henry Doorly Zoo, Omaha NE, USAE-mail address: [email protected] (J. Krebs).

Background: As the cost of antivenom continues to riseand zoo budgets become more restricted it is more difficultto justify the costly expense of antivenom. The number ofcritically endangered species is on the rise and tangible in-situ and ex-situ conservation projects may be the only wayto ensure their sustainability. Though both are criticalcomponents of an institution's collection and mission, theyunfortunately compete for the same funding. As zoomanagers, how do we make the decision as to where wechannel funding and when confronted with that choicehow do we defend it?

Methods: Zoological managers prepare an annualbudget which includes the allocation of funds for thepurchase of antivenom. When venomous reptiles are in thecollection, the antivenom provides safety for the zoo staff,





as well as a broader resource for bites in the community.These same funds could be used to sustain new or estab-lished conservation programs. Some of the factors thatinfluence how the decisions are made include:

� Adherence to the institutional mission statement� Collection diversity and uniqueness� Commitment of the institution to in-situ and ex-situ

conservation projects� Influence on annual attendance provided by

a species� Capabilities of staff that will handle venomous

species� Educational program tie-in� Individual interest of the manager

Results: After all factors are carefully reviewed anddiscussed by managers and staff, the decision whether topurchase antivenom or to fund conservation programsmust be made.

Discussion: Individual philosophical opinions will playa significant role in the process. Institutions havea commitment to the conservation of species but mustgenerate revenue to finance these commitments. A uniqueand diverse collection can ensure continual public financialsupport and help fund conservation work.

Conclusions: Each institution and individual managermust determine the balance within their collection basedon their mission. It will always be a challenge for zoomanagers to determine whether the cost of keepingvenomous species has greater value to the institution thansupporting conservation efforts. The overall value deter-mines how funds are allocated to antivenom costs andconservation support.

Keywords: Cost, antivenom, conservation10.1016/j.toxicon.2012.04.292

292. How Do Komodo Dragons Kill Their Prey?: Lack ofRole for Oral Flora in Predation

Ellie J.C. Goldstein 1,2, Kerrin L. Tyrrell 1, Diane M. Citron 1,Cathleen R. Cox 3, Ian M. Recchio 3, Ben Okimoto 4,Judith Bryja 5, Bryan G. Fry 6

1R. M. Alden Research Laboratory, Culver City, California, USA2UCLA School of Medicine, Los Angeles, California, USA3 Los Angeles Zoo and Botanical Garden, Los Angeles, California, USA4Honolulu Zoo, Honolulu, Hawaii, USA5Houston Zoo, Houston, Texas, USA6Venom Evolution Laboratory, School of Biological Sciences, University ofQueensland, St.Lucia, AustraliaE-mail address: [email protected] (E.J.C. Goldstein).

Background: While the assumption continues tocirculate that the oral flora of the Komodo dragon (Varanuskomodoensis) exerts a lethal effect on its prey, supportivebacteriological evidence is sparse. One previous report wasbased on field observation, while a single subsequent studysuggested Pasteurella multocida as the putative culpritpathogen despite its recovery from only 2/39 wild andcaptive dragons studied.

Methods: A new study of both the aerobic and anaer-obic oral flora of 16 captive Komodo dragons (including 10adults and 6 neonates) using saliva and gingival samplescollected by zoo personnel, inoculated into anaerobictransport media and couriered to a reference laboratoryrevealed different results. Strains were identified by stan-dard methods and 16S rRNA gene sequencing whenrequired. Adult dragons grew 128 unique organismsincluding 37 aerobic gram-negative rods (1–8 per spec-imen); 50 aerobic gram-positive bacteria (2–9 per spec-imen), especially Staphylococcus sciuri and Enterococcusfaecalis, as well as 41 anaerobes (1–6 per specimen),especially clostridia. Hatchlings grew aerobes but notanaerobes. There was some strain variation by zoo, likelyreflective of different dietary components. No virulentspecies were recovered. These data correlate with bacteri-ologic studies of the oral flora of other reptiles and suggestthat V. komodoensis oral flora is simply reflective of the gutand skin flora of their recent meals and environment, and isunlikely to cause rapid fatal infection.

Discussion: This suggests prey capture is largely due tomechanical damage alone for smaller prey, and mechanicaldamage plus venom induced anticoagulation for largerprey. Any wound sepsis to prey such as water buffalo aredue to this animal living outside its natural ecology asa result of introduction by man, with attempted predationby komodo dragons operating outside evolutionary selec-tion pressure. Fatal infection could be due to seeking refugein local feces-laden water holes rather than their nativefresh water pools.

Keywords: Komodo dragon, microbiology, oral flora10.1016/j.toxicon.2012.04.293

293. Coral Snakes, Antivenoms, Hospitals and Zoos:How Can One Little Snake Cause So Many Problems?

Stan MaysHouston Zoo, Inc., Department of Herpetology, Houston, TX, USAE-mail address: [email protected].

Background: The Texas coral snake, Micrurus tener, isthe only elapid snake found in the state of Texas. Althoughthey are relatively common in the Houston area, they areonly rarely encountered, being very shy and secretive intheir habits, and almost never bite. Less than 1% ofvenomous snake bites in the United States are attributed toCoral snakes. However, due to the scarcity of the FDAapproved Wyeth Coral snake antivenom, the Houston Zoooften receives calls requesting antivenom from areahospitals when a Coral snake envenomation occurs insoutheast Texas.

Problem: The Houston Zoo has provided Bioclon Cor-almyn and Instituto Clodomiro Picado Anti Coral anti-venoms to area hospitals in the past upon request.However, there have been numerous problems associatedwith this policy such as antivenom delivery, use, reim-bursement or replacement, return, and accountabilitywhich has resulted in a considerable expenditure of time,money, and angst for the zoo and zoo staff.




Discussion: These difficulties led to the development ofa standardized protocol and associated documentationwith the delivery of Coral snake antivenom to area hospi-tals. The new procedures have, so far, proven successful andhave increased not only accountability (on the part of thehospitals involved) but also zoo willingness and ability toprovide antivenom in a timely and efficient manner whenrequested. These new protocols are now used wheneverthe zoo is requested to provide antivenom for any exoticspecies and not just for Coral snakes.

Keywords: Coral snake, antivenoms, zoos, hospitals10.1016/j.toxicon.2012.04.294

S. Veterinary Toxinology

Fig. 1. Study schematic.

294. A Randomized Multicenter Trial of CrotalidaePolyvalent Immune Fab Antivenom for the Treatment ofRattlesnake Envenomation in Dogs

Michael E. Peterson 1,2, Michael Matz 3, Karen E. Seibold 4,Signe Plunkett 5, Scott Johnson 6, Kevin Fitzgerald 7

1Reid Veterinary Hospital, Albany, OR, USA2VIPER Institute, University of Arizona, Tucson, AZ USA3Veterinary Specialty Center of Tucson, Tucson, AZ, USA4Animal Urgent Care and Specialty Group, Escondido, CA, USA5Animal Health Services, Cave Creek, AZ, USA6 Emergency Animal Hospital of Northwest Austin, Austin, TX, USA7VCA Alameda East Veterinary Hospital, Denver, CO, USAE-mail address: [email protected] (M.E. Peterson).

Objective: To determine clinical efficacy of the Crotali-dae polyvalent immune Fab (ovine) (Crofab�) antivenom(OPAC) against progressive Crotalid envenomation in thedog as reflected in stabilization or improvement of snake-bite severity scores (SSS). Additionally, due to the potentialdecreased half life of the Fab antibodies in dogs wecompared SSS between dogs receiving two different dosingregimes.

Design: Prospective, clinical trial.Setting: Five veterinary emergency and critical care

facilities one in each city Tucson, Az; Phoenix, Az; Austin,Tx; Escondido, Ca; and Denver, Co .

Animals: 115 client owned Crotaline (rattlesnake)snake-bitten dogs in whom worsening of the envenoma-tion syndrome was observed before OPAC treatment.

Interventions: In a multicenter randomized clinicaltrial a single dose (1 vial) of OPCA alonewas comparedwithtwo doses (1/2 vial each) administered 6 hours apart.Standard supportive care was provided in all cases.

Measurements and Main Results: Data was availablefor 115 patients, nine of which were fatalities. All patients’clinical conditionwas documented with a standardized SSSsystem accounting for each major body system. Eachfatality received maximum severity scores of 20. The meanseverity score of the 115 patients decreased from 4.19 to3.29 points and there was no difference between the twotreatment groups. The mean severity score of the 107patients without fatalities decreased from 4.16 to 2.15.Antivenom related acute reactions occurred in 6 patients

(6%), and no serum sickness occurred within the 95 casescontacted at the two week post treatment follow up.

Conclusions: In the first canine randomized trial ofantivenin in the United States, Fab AV effectively stabilizedor terminated venom effects. There were no statisticaldifferences between treatment groups within the studytime frame.

Keywords: Antivenom, snakebite, dogs10.1016/j.toxicon.2012.04.295

295. F(ab’)2 Antivenom in Dogs Envenomated by PitVipers

Karen E. Seibold 1, Craig W. Woods 2, Raegan J. Wells 3

1Animal Urgent Care and Specialty Group, Escondido, CA, USA2BioVeteria Life Sciences, LLC, Prescott, AZ, USA3 Emergency Animal Clinic, PLC, Phoenix, AZ, USAE-mail address: [email protected] (C.W. Woods).

Background: A previous clinical study using a F(ab’)2antivenom was performed in pit viper envenomation indogs. Some of these datawere previously presented at 2010International Veterinary Emergency and Critical CareSymposium. Pit viper envenomation in dogs is a commonsituation and represents an excellent comparative clinicalmodel to study the effects of antivenoms owing to clinical




pathology, natural envenomation, and diagnostic capturemethods.

Methods: This review is from a prospective study in 74dogs presenting at 10 veterinary emergency hospitals inArizona, Southern California, Texas and Florida whichwere naturally envenomated by pit vipers. Dogs wereenrolled based on inclusion and exclusion criteria andwere randomized into two F(ab’)2 antivenom treatmentgroups; Group A (n¼37) received 4 vials at baseline andGroup B (n¼37) received 2 vials at baseline and 2 vials at 3hours. Clinical scores and blood were collected at baseline(pre-treatment) and at hours 1, 3, 6, 12 and 24 aftertreatment.

Results: 73 of 74 dogs survived, with one small dog(<7kgs) succumbing to an intra-ocular envenomation. Overtime, all dogs (Groups A and B) had statistically significantdecreases in pain (p<0.001), extension of pain (p<0.001),discharge amount (p<0.001), discharge type (p<0.001),and overall clinical morbidity scores (p<0.001). Bothgroups had significant improvements in prothrombin time(PT) (p<0.005), and echinocytosis (p<0.001). Group A hadsignificant reductions (p¼0.025) in partial thromboplastintime (PTT) and Group B dogs had significant improvement(p¼0.016) in platelet counts over time.

Discussion: The use of pit viper antivenom in veterinarymedicine is popular as the propensity for envenomation isremarkably higher than for human medicine. In veterinarymedicine, the use of F(ab’)2 antivenoms appears to haveconsiderable advantages compared to whole IgG or Fabproducts, primarily related to affordability, ease of use,handling, andpreferred distribution and eliminationprofiles.

Conclusions: This study demonstrated that adminis-tration of a F(ab’)2 pit viper antivenom is well tolerated andassociated with a significant improvement in various clin-ical and hematologic abnormalities associated with pitviper envenomation.

Keywords: Antivenom, veterinary, dogs10.1016/j.toxicon.2012.04.296

296. Crotalidae Venom Levels in Dogs Before and AfterAdministration of F(ab’)2 Antivenom

Craig W. Woods 1, Karen E. Seibold 2, Raegan J. Wells 3

1BioVeteria Life Sciences, LLC, Prescott, AZ, USA2Animal Urgent Care and Specialty Group, Escondido, CA, USA3 Emergency Animal Clinic, PLC, Phoenix, AZ, USAE-mail address: [email protected] (C.W. Woods).

Background: Some of these data were previously pre-sented at 2011 International Veterinary Emergency andCritical Care Symposium. The dog is an excellent compar-ative model to study envenomation owing to their simi-larity to human envenomation. Serum venom levelsprovide information to assess the neutralization propertiesof antivenoms.

Methods: This study evaluated the venom neutralizingeffects of a F(ab’)2 antivenom (Instituto Bioclon) in 55 dogsnaturally envenomed by pit vipers. Dogs received 4 vials ofantivenom in the first 3 hours and serum was collected pre-

treatment and at 1, 3, 6, 12, and 24 hours after antivenomadministration. Venom levels (ng/mL)weredeterminedusingan enzyme-linked immunosorbent assay. Statistical analysiswas performed using 1-way ANOVA repeated measures.

Results: The average venom level before antivenomadministration was 158 þ/- 22.3 ng/mL; average venomlevels were significantly decreased (p<0.001) at T1 (0.07þ/- 0.07 ng/mL), T3 (0.12 þ/- 0.11 ng/mL), T6 (0.00 ng/mL),T12 (1.74 þ/- 0.81 ng/mL), and T24 (5.09 þ/- 1.67 ng/mL).

Discussion: Venom level measurements are clinicallyvaluable for a variety of reasons. First, venom levels canconfirm envenomation. Serum venom level measurementscan determine if circulating venom was neutralized byantivenom and if more antivenom is required. In addition,venom levels are valuable in understanding venom andantivenom pharmacokinetics.

Conclusions: This study demonstrates that adminis-tration of F(ab’)2 pit viper antivenom is associated witha rapid and prolonged neutralization of circulating pit vipervenoms.

Keywords: Venom, dogs, antivenom10.1016/j.toxicon.2012.04.297

297. A Retrospective Review of Coral SnakeEnvenomation in the Dog and Cat: 20 cases 1996 to 2011

Mayrim L. Pérez, Karlie J. Fox, Michael SchaerDepartment of Small Animal Clinical Sciences, College of VeterinaryMedicine, University of Florida, Gainesville, FL, USAE-mail address: [email protected] (M.L. Pérez).

Objective: To expand the current limited data base anddescribe clinical signs, treatment, and outcomes of dogsand cats after envenomation byMicrurus fulvius fulvius andto report our clinical experience with the use of Coralmyn.

Animals: 16 dogs and 4 cats withMicrurus fulvius fulviusencounter or envenomation evaluated at a universityteaching hospital.

Methods: Retrospective study.Results: Medical records meeting the inclusion criteria

were reviewed and evaluated for signalment, date andtime of the snake encounter, elapsed time betweenencounter and hospital examination, initial physicalexamination results, antivenom type, length of hospitali-zation, and outcome. Initial physical examination findingsincluded: quiet mentation, tetraparesis, ptyalism,tachypnea, shallow breathing, decreased to absent gagreflex, ataxia, muscle fasciculations, and decreased spinalreflexes. Laboratory findings in dogs included proteinuria,bilirubinuria, hemeproteinuria, increased AST, increasedALT, and hemolysis. Four dogs and 2 cats received Cor-almyn and 4 dogs received North American Coral SnakeAntivenom (NACSA). Adverse reaction to antivenom wassuspected in 1 dog that received NACSA. Eight of 11envenomated dogs were survivors with a median lengthof hospitalization of 4.5 days. Two of 3 envenomated catswere survivors with a median length of hospitalization of4 days. Two dogs were euthanized, 1 dog suffered respi-ratory arrest, and 1 cat developed tachycardia that




progressed to pulseless electrical activity. Five dogs and 1cat in the encounter group survived to discharge.

Conclusions: Micrurus fulvius fulvius envenomationis likely in the dog that is found to have concomitant lowermotor neuron neuropathy, bulbar palsy, and hemolysis.Early diagnosis is crucial as antivenom administration canreduce morbidity and perhaps mortality. Prognosis is

considered good with 71% of the envenomated patients inthis study surviving to discharge. Supportive care whichmay include ventilator assistance and antivenom admin-istration are the mainstays of therapy.

Keywords: Micrurus fulvius fulvius, Coralmyn, Veterinary toxinology10.1016/j.toxicon.2012.04.298

17th world congress of the international society on

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