Abstracts Toxins 2012 / Toxicon 60 (2012) 95–248 199

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: mrlsando@butantan.gov.br (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: mospadafora@butantan.gov.br (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

Abstracts Toxins 2012 / Toxicon 60 (2012) 95–248200

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: juri.siigur@kbfi.ee (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: am.tibabuzo205@uniandes.edu.co (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