ISSN 0073 - 9901 MIBUAH GOVERNO DO ESTADO DE SÃO PAULO SECRETARIA DE ESTADO DA SAÚDE COORDENAÇÃO DOS INSTITUTOS DE PESQUISA INSTITUTO BUTANTAN SÃO PAULO, SP - BRASIL Memórias do Instituto Butantan VOLUME 52, SUPLEMENTO, 1990 cm 2 3 4 5 6 'SCÍELOq 2.1 12 13 14 15 16 As "MEMÓRIAS DO INSTITUTO BUTANTAN” têm por finalidade a apre¬ sentação de trabalhos originais que contribuam para o progresso nos campos das Ciências Biológicas, Médicas e Químicas, elaborados por especialistas nacionais e estrangeiros. São publicadas sob a orientação da Comissão Editorial, sendo que os con¬ ceitos emitidos são de inteira responsabilidade dos autores. The "MEMÓRIAS DO INSTITUTO BUTANTAN" are the vehicle of com- munication for original papers written by national and foreign specialists who contribute to the progress of Biological, Medicai and Chemical Sciences. They are published under the direction of the Editorial Board which assu¬ mes no responsability for statements and opinions advanced by contributors. Diretor do Instituto Butantan Dr. Willy Beçak Comissão Editorial Henrique Moisés Canter — Presidente Adolpho Brunner Júnior — Membros Olga Bohomoletz Henriques Raymond Zelnik Sylvia Lucas Denise Maria Mariotti — Bibliotecária Indexado/lndexed: Biosis Data Base, Current Contents, Index Medicus. Periodicidade: irregular Permuta/Exchange: são feitas entre entidades governamentais, com publicações congêneres, mediante consulta prévia. Exchanges with similar publications can be settled with academic and governmental institutions through prior mutual agreement. Endereço/Address. Instituto Butantan — Biblioteca. Av. Vital Brasil, 1.500 05504 — São Paulo, SP — Brasil Telefone/Telephone: (011)813-7222 - R. 129 - Telex: (011) 83325 BUTA-BR Telefax: (011) 815-1505 cm 2 3 4 5 6 SCÍELO 10 n 12 13 14 15 16 Governo do Estado de São Paulo Secretaria de Estado da Saúde Coordenação dos Institutos de Pesquisa Instituto Butantan — São Paulo — SP — Brasil MEMÓRIAS DO INSTITUTO BUTANTAN Volume 52, Suplemento, 1990 I SIMPÓSIO DA SOCIEDADE BRASILEIRA DE TOXINOLOGIA "PEÇONHAS E ENVENENAMENTO NO BRASIL" À Memória de Gastão Rosenfeld (1912-1990) 5-7 setembro 1 990 Instituto Butantan São Paulo-Brasil Publicação patrocinada pela Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) São Paulo, SP — Brasil 1990 MEMÓRIAS DO INSTITUTO BUTANTAN. (Secretaria de Estado da Saúde) São Paulo, SP — Brasil, 1918 — 1918 - 1983/84, 1-47/48 Publicação interrompida de 1985 a 1986. 1987, 49(1-3) 1988, 50(1-3, supl.) 1989, 510-4) 1990, 520-3, supl.) ISSN 0073-9901 MIBUAH CDD 614.07205 Solicita-se permuta/Exchange desired 10 11 12 13 14 SIMPÓSIO DA SOCIEDADE BRASILEIRA DE TOXINOLOGIA "PEÇONHAS E ENVENENAMENTO NO BRASIL" DIRETORIA DA SBTx (1988-1990) Presidente: Oswaldo Vital Brazil Vice-Presidente: Júlia Prado-Franceschi Secretária Geral: Eva Maria A. Kelen Tesoureiro: Antônio Carlos Oliveira COMISSÃO ORGANIZADORA DO A Diretoria da SBTx João Luiz Costa Cardoso Vera F. Pires de Campos Flenrique Moisés Canter SIMPOSIO DA SBTx ASSESSORIA DE INFORMÁTICA Miguel Casella Júnior AGRADECIMENTOS Instituto Butantan UNICAMP FAPESP Johnson & Johnson Banespa cm 'SciELO 0 11 12 13 14 15 16 I Simpósio da Sociedade Brasileira de Toxinologia Instituto Butantan, 5 a 7 de setembro de 1990 À Memória de GASTÃO ROSENFELD Simpósio sobre: "Peçonhas e Envenenamento no Brasil" PROGRAMA 5/9/90 - 4? feira 9:00 Abertura 9:20 Veneno Crotálico Presidente: Carlos J. 1 Laure (Ribeirão Preto — SP) Aspectos bioquímicos — 20 minutos Relator: Carlos J. Laure, Dep. Bioquímica, Fac. Medicina, Ribei¬ rão Preto, USP 2 — Farmacologia: Crotoxina — 20 minutos Relator: Barbara E. Flawgood (Londres) 3 — Farmacologia: Crotamina — 20 minutos Relator: Oswaldo Vital Brazil, Dep. Farmacologia, Unicamp 10:20 Intervalo — 20 minutos 10:40 4 — Farmacologia: Giroxina e Convulxina (Filme) — 20 minutos Relator: Júlia Prado-Franceschi, Dep. Farmacologia, Unicamp 11:00 Discussão — 1 hora 12:30 Apresentação de painéis — 1 hora 14:00 Ofidismo I Presidente: Cyro Coimbra de Rezende (Brasília-DF) 1 — Epidemiologia do Ofidismo — 20 minutos Relator: Lindioneza A. Ribeiro, Hospital Vital Brazil 2 — Acidente Crotálico — 40 minutos 2.1 Aspectos gerais do envenenamento 2.2 Aspectos clínicos especiais Coordenadora: Mariza M. Azevedo-Marques, USP, Ribeirão Preto Participantes: Carlos Faria Santos Amaral (UFMG), Palmira Cupo (HC, USP-RP). 15:00 Discussão — 1 hora 16:00 Intervalo — 20 minutos 16:20 Ofidismo II — Veneno e Envenenamento Elapídico — 40 minutos Presidente: José Ronan Vieira (Campinas — SP) 1 — Veneno Elapídico: Farmacologia Relator: Oswaldo Vital Brazil, Dep. Farmacologia, Unicamp 2 — Clínica de Envenenamento Elapídico Relator: Fábio Bucaretchi, Unicamp 17:00 Discussão — 1 hora 6/9/90 — 5? feira 9:20 Veneno Botrópico Presidente: Léa Rodrigues Simioni (Campinas — SP) 1 — Aspectos Bioquímicos — 20 minutos Relator: Fajga R. Mandelbaum, Serv. Bioquímica, IB 2 — Farmacologia: Alterações locais — 20 minutos Relator: José Maria Gutierrez, Inst. Clodomiro Picado, Costa Rica 3 — Farmacologia: Alterações hematológicas — 20 minutos Relator: Ida S. Sano-Martins, Seção Hematologia, IB cm 'SciELO 0 11 12 13 14 15 16 10:20 Intervalo — 20 minutos 10:40 4 — Farmacologia: Alterações cardiovasculares — 20 minutos Relator: Júlia Prado-Franceschi, Dep. Farmacologia, Unicamp 11:00 Discussão — 1 hora 12:30 Apresentação de painéis — 1 hora 14:00 Ofidismo III — Acidente Botrópico — 40 minutos Presidente: Wilmar Dias da Silva (São Paulo — SP) 1 — Aspectos gerais do envenenamento 2 — Aspectos regionais Coordenador: João Luiz Costa Cardoso, Plospital Vital Brazil Participantes: João Aris Kouyoumdjian, São José do Rio Preto, e Raimundo Nonato Leite Pinto, Goiânia 14:40 Discussão — 1 hora 15:40 Intervalo — 20 minutos 16:00 Assembléia Geral da SBTx 7/9/90 - 6? feira 9:20 Veneno de Artrópodos Presidente: Alexandre Pinto Corrado (Ribeirão Preto — SP) 1 — Aspectos químicos e farmacológicos de venenos de escorpião e aranha — 20 minutos Relator: Carlos R. Diniz, Dep. Bioquímica, UFMG, Belo Horizonte 2 — Farmacologia do veneno de escorpião — 20 minutos Relator: Lineu Freire-Maia, Dep. Farmacologia, UFMG, Belo Ho¬ rizonte 3 — Farmacologia do veneno de Phoneutria — 20 minutos Relator: Marcos Dias Fontana, Dep. Farmacologia, Unicamp, Campinas 10:20 Intervalo — 20 minutos 4 — Farmacologia do veneno de Loxosceles — 20 minutos Relator: João Luiz Costa Cardoso, Hospital Vital Brazil 11:00 Discussão — 1 hora 12:30 Apresentação de painéis — 1 hora 14:00 Escorpionismo Presidente: Lineu Freire-Maia (Belo Horizonte, MG) 1 — Ação do veneno de escorpião sobre a musculatura cardíaca Estudo clínico e laboratorial — 10 minutos 2 — Clínica do escorpionismo — 30 minutos Coordenador: José Américo de Campos, Belo Horizonte Participantes: Palmira Cupo, Sylvia Hering, Mariza M. Azevedo- -Marques (HC, Ribeirão Preto, USP) 14:40 Discussão — 1 hora 15:40 Intervalo — 20 minutos 16:00 Araneísmo Presidente: Carlos R. Diniz (Belo Horizonte, MG) 1 — Acidentes por Phoneutria — 20 minutos Relator: Fábio Bucaretchi, Unicamp, Campinas, SP 2 — Acidentes por Loxosceles — 20 minutos Relator: Francisco O.S. França, Hospital Vital Brazil, IB, São Paulo 16:40 Discussão — 1 hora 17:40 Intervalo — 20 minutos 18:00 Encerramento 20:00 Jantar de encerramento cm SciELO 10 11 12 13 14 15 16 Mem. Inst. Butantan, 52 (supl.), 1990 FIRST SYMPOSIUM OF THE BRAZILIAN SOCIETY OF TOXINOLOGY: ON VE- NOMS AND ENVENOMING CAUSED BY VENOMOUS ANIMALS IN BRAZIL. In memory of Gastão Rosenfeld (1912-1990), and as one of the commemora- tive events of the 90 years of Instituto Butantan foundation (1901-1991) São Paulo, 5-7 September, 1990. I. Epidemiology of snakebite accidents in Brazil. II. Biochemistry and pharmacology of the South American rattlesnake venoms. III. Human accidents caused by the South American rattlesnake. IV. Coral snakes: Pharmacology of venoms and accidents. V. Biochemistry and pharmacology of Bothrops snake venoms. VI. Human accidents caused by Bothrops snakes. VII. Biochemistry and pharmacology of scorpion and spider venoms. VIII. Human accidents caused by scorpions and spiders. 7 cm 2 3 z 5 6 11 12 13 14 15 16 Mem. Inst. Butantan, 52 (supl.), 1990 CONTENTS 1. Oswaldo Vital Brazil 2. Willy Beçak 3. Lindioneza Adriano Ribeiro 4. Carlos Julio Laure 5. Barbara E. Hawgood 6. Oswaldo Vital Brazil 7. Júlia Prado-Franceschi 8. Mariza M. Azevedo- -Marques, Palmira Cupo, Carlos Faria S. Amaral and Sylvia Hering 9. Oswaldo Vital Brazil 10. Fábio Bucaretchi 11. Fajga R. Mandelbaum 12. José Maria Gutierrez 13. Ida S. Sano-Martins 14. Júlia Prado-Franceschi 15. João Luís C. Cardoso 16. João Aris Kouyoumdjian 17. Raimundo Nonato L. Pinto 18. Carlos Ribeiro Diniz 19. Lineu Freire-Maia 20. Marcos Dias Fontana 21. Fábio Bucaretchi 22. Francisco O.S. França POSTERS ABSTRACTS AUTHORS INDEX I - PLENARY SESSIONS II - POSTERS SESSION PRESENTATION 11 ABERTURA DO SIMPÓSIO 13 EPIDEMIOLOGY OF OPHIDIC ACCIDENTS 15 CROTALUS DURISSUS TERRIFICUS VENOM: CROTOXIN AND INTER-CRO 17 CROTOXIN, THE PHOSPHOLIPASE A 2 NEUROTOXIN FROM THE VENOM OF CROTALUS DURISSUS TERRIFICUS 21 PHARMACOLOGY OF CROTAMINE 23 ON THE PHARMACOLOGY OF CON- VULXIN AND GYROXIN 25 RATTLESNAKE BITES. CLINICAL FEA- TURES AND COMPLEMENTARY TESTS 27 PHARMACOLOGY OF CORAL SNAKE VENOMS 31 ELAPIDIC ENVENOMATION: CLINICAL FEATURES 33 SNAKE VENOM HEMORRHAGINS 35 LOCAL PATHOLOGICAL EFFECTS INDUCED BY BOTHROPS SNAKE VENOMS 37 HEMATOLOGICAL DISTURBANCES INDUCED BY BOTHROPS VENOM 39 SYSTEMIC ACTIVITIES OF BOTHROPIC VENOMS 41 BOTHROPIC ACCIDENTS 43 INTRACRANIAL HAEMORRAGE AFTER SNAKE BITE 45 SNAKEBITE ACCIDENTS IN GOIÁS 47 CHEMICAL AND PHARMACOLOGICAL PROPERTIES OF SCORPION AND SPIDER VENOMS 49 PHARMACOLOGY OF TITYUS SERRULATUS SCORPION VENOM 51 PHARMACOLOGY OF PHONEUTRIA VENOM 59 ACCIDENTS WITH SPIDERS OF THE PHONEUTRIA GENUS 61 ACCIDENTS BY LOXOSCELES 63 cm 'SciELO 0 11 12 13 14 15 16 Mem. Inst. Butantan, 52 (supl.), 1990 PRESENTATION During the VII Annual Symposium of The Academia de Ciências do Estado de São Paulo (ACIESP) on Proteinous Toxins, which was held in April of 1988 at the Universidade de Campinas (UNICAMP), a group of scientists decided to found a society that would bring together biochemists, pharmacologists, immunologists and pathologists interested in investigations concerning toxins of animal, plant or microbial origin, as well as clinicians engaged in studies of accidents caused by venomous animais. The Brazilian Toxinology Society was inaugurated in November of that year, and now held its first Symposium. The aim of the Or- ganizing Committee was to allow the integration at this meeting of the advances made in the past few years in the knowledge of basic aspects of venom and their toxins, especially of Brazilian species, and those acquired in the clinicai area of accidents caused by venomous snakes, scorpions and spiders. Scientists and cli¬ nicians participating in the Symposium have much gained, I believe, from this association of presentation of knowledge in basic and clinicai areas. The com¬ mittee decided to reserve a time for the discussion of the themes at the end of each group of presentations, making it possible for any member of the Symposi¬ um to comment the lectures, to request clarifications, and eventually to present his or her experimental or clinicai contributions to the theme being discussed. At the suggestion of some members, a session for the presentation of posters was created. We note with satisfaction, the presentation of more than forty Com¬ munications in the form of posters demonstrating the considerable activity of var- ious research groups in our country in the field of toxinology. In July of the current year, everyone who knew him personally, or through his publications, received the distressing news of the death of Gastão Rosenfeld. In view of his important experimental and clinicai contributions to Brazilian toxi¬ nology, the Organizing Committee decided to honor him posthumously by dedicat- ing the First Symposium of the Brazilian Toxinology Society to his memory. In this issue of the Memórias do Instituto Butantan, two fuil-length papers and twenty abstracts of invited presentations are published as well as forty nine ab- stracts of pôster Communications. I hope the reader will appreciate this volume which is being published thanks to the cooperation of the authors, the agree- ment of Professor Willy Beçak, Director of Instituto Butantan and Dr. Henrique Moisés Canter, Chief Editor of the Mem. Inst. Butantan, as well as the financial support of the Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP). I am deeply grateful to all of them. Campinas, October, 1990. O. VITAL BRAZIL 11 cm 2 3 z 5 6 11 12 13 14 15 16 Mem. Inst. Butantan, 52 (supl.l, 1990 ABERTURA DO SIMPOSIO A realização pela Sociedade Brasileira de Toxinologia do seu 1 0 Simpósio sobre Peçonhas e Envenenamento no Brasil, no Instituto Butantan, tem um cu¬ nho histórico importante. O Instituto Butantan, desde asua fundação há 90 anos tem dado importantes contribuições e avanços no conhecimento das peçonhas e no tratamento dos acidentes por serpentes, aranhas e escorpiões. Esta saga iniciada por Vital Brazil foi continuada por vários seguidores, notabilizando-se, em época mais recente, Gastão Rosenfeld, pela sua contribuição teórica e práti¬ ca, que deixou escola. Desde a descoberta da Bradicinina, juntamente com Mau¬ rício Rocha e Silva e Wilson Beraldo da Silva, seus estudos em Hematologia, até a implantação do hospital e padronização do tratamento de acidentes por animais peçonhentos, dedicou esse cientista sua vida à pesquisa e à medicina. É, pois, com grande satisfação que fazemos a abertura desse conclave, na instituição que é o berço da Toxinologia no Brasil como homenagem à memória de Gastão Rosenfeld, que foi um dos seus grandes expoentes. Trabalhos de relevo serão apresentados e discutidos neste Simpósio, con¬ tribuindo para o melhor conhecimento dessa importante disciplina. No Brasil, abun¬ dante em fauna e flora, a toxinologia tem grande interesse, não só na clínica médica, como também pelo potencial que ainda resta para ser investigado e des¬ coberto. Como Diretor, em nome do Instituto Butantan e como cientista, queremos expressar a nossa satisfação em receber os colegas e os convidados da Sociedade Brasileira de Toxinologia em nossa casa. Auguramos muito sucesso à reunião que ora se inicia, sob a presidência desse renomado pesquisador, que é o Prof. Oswaldo Vital Brazil, no âmbito das comemorações do nonagésimo aniversário do Instituto Butantan. W. BEÇAK Diretor do Instituto Butantan 13 cm 2 3 L 5 6 11 12 13 14 15 16 Mem. Inst. Butantan, 52 (supl.), 1990 EPIDEMIOLOGY OF OPHIDIC ACCIDENTS Lindioneza Adriano Ribeiro, Hospital Vital Brazil — Instituto Butantan, São Paulo, SP. Venomous Brazilian snakes belong to four genera: Bothrops, Crotalus, Lache- sis and Micrurus. Bothrops and Micrurus occur in the whole national territory, Lachesis in humid areas, in the forest zone and the Amazonian Region, whilst, in contrast Crotalus is not found in these regions and the littoral, prefering dry areas. 2 ' 3 At the beginning of this century. Vital Brazil, founder and Director of the Insti- tute Butantan, called attention to the importance of ophidic accidents as a Public Health problem. The number of accidents were then evaluated as 19,200 each year, being principally due to the ''jararaca'' (Bothrops)'. For the year 1987, 21,463 cases, including 123 deaths were notified to the Department of Health. The incidence calculated for Brazil was 1 5.5/100,000 inhabitants and the death- rate of 0.6% showed a variation within the macroregions from 0.3% (Southeast) to 2.1% (North). 0f the 20,884 notifications of accidents received from June 1986 to December 1987 that included information on the genus of the snake, 88.3% were caused by Bothrops, 8.3% by Crotalus, 2.7% by Lachesis and 0.7% by Micrurus. The greater number of deaths caused by snakes of the genus Bothrops followed by Crotalus, may be due to the frequency of their presence, in as much as the deadliness of bothropic accidents (0.5%) is less than that of crotalic ones (3.3%) 6 . In the State of São Paulo, for the years 1986, 1987 and 1988, 6,308 acci¬ dents were notified (incidence of 6.8/100,000 inhabitants) and of 4,685 notifi¬ cations with reference to the genus of the snake, 85.3% were by Bothrops, 13.6% by Crotalus and 1.1% by Micrurus. Lethality was 0.3%. 11 Accidents depend on the interrelationship between snakes, environment and man. Bites by Bothrops and Crotalus, occur on the inferior limbs in 75% 4 and 71 % of the cases respectively. Snakes of the genus Micrurus are of subterrane- an habit 2 and in general bites are on the hand as a result of handling. 8 Veno¬ mous snakes are predominantly nocturnal in habit, 2 including Bothrops jararaca.' 3 However accidents occur more frequently during daytime 9 the time 15 cm 2 3 L 5 6 11 12 13 14 15 16 Mem. Inst. Butantan, 52 (supl.), 1990 of man's highest activity. Moreover, bites mostly occur in the hot months of the year, 12 when the snakes are more active 13 and within the rural zone 1 where agriculture is not mechanized. Other factors may contribute to the incidence of accidents. Human habitation may lead to the proliferation of rodents, thus at- tracting the snakes 2 . Extermination of non venomous snakes may eliminate the natural predators as well as lessen the competition for food and thus lead to an increase in the number of venomous snakes. Due to social, economic, cultural and climatic conditions, the rural labourer usually works without adequate pro- tective clothing. 1 ' 5 Among the recommended measures for the prevention of accidents, 15 is the use during the work in the field of long-leg boots or puttees, and gloves of leather shavings. A simple boot that covers the ankle may prevení about 60% of the accidents and puttees, another 10%. However, in field labourer using long-leg boots or puttees, accidents tend to be the most severe, being frequently caused by snakes of great dimensions. 5 ' 6 ' 10 REFERENCES 1. BRAZIL, V. A defesa contra o ophidismo. São Paulo, Pocai & Weis, 1911. 152p. 2. FONSECA, F. Animais peçonhentos. São Paulo, Empresa Gráfica da "Revista dos Tribunais", 1949. 376p. 3. FIOGE, A. R. & ROMANO-FIOGE, S.A.R.W.L. Sinopse das serpentes peçonhentas do Brasil. 2. ed. Mem. Inst. Butantan, 42/43: 373-499, 1978/79. 4. JORGE, M. T.; RIBEIRO, L.A.; MENDONÇA, J. S. Acidente botrópico: experiência del Hospital Vital Brazil. In: CONGRESO 5Ò ANIVERSARIO DEL INSTITUTO DE MEDI¬ CINA TROPICAL "PEDRO KOURI". Resumenes. Havana, Cuba, 1988. p. 189-190. 5. JORGE, M.T. & RIBEIRO, L.A. Acidentes por animais peçonhentos. In: AMATO-NETO, V. & BALDY, J.L.S. ed. Doenças transmissíveis. 3. ed. São Paulo, Sarvier, 1989, p. 133, 141. 6. RESENDE, C.C.; ARAÚJO, F.A.A.; SALLENAVE, R.N.U.R. Análise epidemiológica dos acidentes ofídicos. Brasília, Ministério da Saúde, Secretaria Nacional de Ações Bᬠsicas de Saúde. 1989. p. 37. 7. RIBEIRO, L.A.; JORGE, M.T.; PIESCO, R.V. Acidente crotálico: experiência do Hospi¬ tal Vital Brazil-lnstituto Butantan. In: CONGRESSO DA SOCIEDADE BRASILEIRA DE MEDICINA TROPICAL, 20 e CONGRESSO DA SOCIEDADE LATINO-AMERICANA DE MEDICINA TROPICAL, 1. Resumos. Salvador, 1984. p. 138. 8. RIBEIRO, L.A. & JORGE, M.T. Acidentes por serpentes do gênero Mícrurus ("Coral") — Análise de sete casos. Rev. Soc. bras. Med. Trop., 19 (supl.): 28, 1986. 9. RIBEIRO, L.A & JORGE, M.T. Alteração do tempo de coagulação sanguínea em pa¬ cientes picados por serpente Bothrops jararaca adulta e filhote. Rev. Hosp. Clin. Fac. Med. S. Paulo, 44(4): 143-145, 1989. 10. RIBEIRO, L. A. & JORGE, M. T. Fatores prognósticos em acidentes por serpentes do gênero Bothrops. In: CONGRESSO DA SOCIEDADE BRASILEIRA DE MEDICINA TRO¬ PICAL, 21. Resumos. São Paulo, 1985. p. 28. 11. RIBEIRO, L. A., PIRES DE CAMPOS, V.A.F.; ALBUQUERQUE, M.J.; TAKAOKA, N.Y. Epidemiological and clinicai aspects of accidents due to poisonous snakes in the State of São Paulo, Brazil, from 1986 to 1988. Toxicon, 28(6): 621, 1990. 12. ROSENFELD, G. Animais peçonhentos e tóxicos do Brasil. In: LACAZ, C.S.; BARUZ- Zl, R.G.; SIQUEIRA JR., W., ed. Introdução à geografia médica do Brasil. São Pau¬ lo, Editora da Universidade de São Paulo, 1972. p. 430-475. 13. SAZIMA, I. Um estudo de biologia comportamental da jararaca, Bothrops jararaca, com uso de marcas naturais. Mem. Inst. Butantan, 50( 31:83-89, 1988. í, i SciELO 16 Mem. Inst. Butantan, 52 (supl.l, 1990 CROTALUS DURISSUS TERRIFICUS VENOM: CROTOXIN AND INTER-CRO Carlos Julio Laure, Department of Biochemistry, Faculty of Medicine of Ribeirão Preto, University of São Paulo. Crotoxin from Crotalus durissus terrificus represents almost 50% of the dry weight of the venom and is the main toxic component of the latter. 6 Crotoxin was separated into two fractions by two different methods, using carboxymethyl- cellulose and DEAE-celiuIose chromatography: 15 ' 9 an acid, nontoxic fraction denoted Crotapotin, and a basic, low toxicity fraction with enzymatic activity denot- ed Phospholipase A 2 . Crotapotin (acid) potentiates the toxicity of phospholipase A (basic) in vivo but inhibits its hemolytic activity in vitro. Both laboratories showed that Crotoxin is a complex formed by the strong as- sociation of crotapotin with phospholipase A 2 . The recombination of these frac¬ tions restores the toxicity of crotoxin. Phospholipase A 2 has a molecular weight of 15,000 daltons, with a P.l. of 8.6. 15 ' 10 lt consists of a single polypeptide chain of 133 residues. 8 The acid component crotapotin has a P.l. of 3.4 and a molecu¬ lar weight of 9,000 daltons according to Horst 10 , and a P.l. of 3.7 according to Breithaupt, 5 who, however, determined the same molecular weight. Breithaupt 5 reduced crotapotin with/3-mercaptoethanol foilowed by carboxyamide methyla- tion and separated three peptide chains linked by disulfide bridges: Chain A with 40 amino acid residues, MW 4,300 Chain B with 34 amino acid residues, MW 3,700 Chain C with 14 amino acid residues, MW 1,600 The N-terminal of the peptide chains only supplies serine for chain A, indicat- ing that the N-terminals of chains B and C are blocked. 5 Crotoxin at high doses acts on the post-synaptic membranes, stabilizing the acetylcholine receptors. 3 ' 17 . Crotapotin only potentiates the toxicity of crotalic phospholipase A, a fact that could be interpreted as a pharmacokinetic phenomenon. This acid fraction can protect phospholipase A against inactivation by binding to low affinity sites, thus preserving it for high-affinity receptors. There is no evidence of in vivo formation of the crotoxin complex. Jeng 11 and Bon 2 proposed a model which they called "chaperon” effect for crotapotin, 17 cm 2 3 z 5 6 11 12 13 14 15 16 Mem. Inst. Butantan, 52 (sup!.), 1990 which is considered to be a phospholipase A "transporter” to the specific site of the appropriate receptor. Using a totally different process, Laure 13 obtained crotapotin from crotoxin in order to determine its primary structure by the following methodology: crotoxin was isolated by filtration on Sephadex G-75 with 0.05 M ammonium formiate, pH 3.5, dialyzed and lyophilized. Crotapotin and phospholipase A 2 were isolat¬ ed on SP-Sephadex C-25 with 0.8 M to 3.0 M guanidine chloride, pH 4.2. After dialysis and lyophilization, crotapotin was filtered on superfine Sephadex G-50 with 0.1 M NH 4 HC0 3) pH 8.2. Crotapotin was tested for purity by amino acid analysis, polyacrylamide gel electrophoresis, N-terminal on polyamide plates by dansylation and then reduced with dithiothreitol and carboxymethylated with iodoacetic acid by standard methods, followed by desalting on Sephadex G-10 and lyophilization. The lyophi¬ lized product was submitted to filtration on Sephadex G-50 S.F. with 0.1 M NH 4 HC0 3 , pH 8.2, and three peaks corresponding to chains A, B and C were ob¬ tained. Chain C was isolated and sequenced by Edman-dansyl and dabitization, in col- laboration with my graduate student Gabilan. Chain B was isolated and cleaved with BrCN and peptides I and II were isolated by filtration on Sephadex G-50 S.F. Sequencing was done by my post-graduate student Conti using the same process as for chain C. Chain A was submitted to different enzymatic treatments, the pep¬ tides were isolated and purified and sequenced by the Edman-Dansyl method. Using sequencing via Edman-Dansyl and dabitization, the primary structures of chains A, B and C of crotapotin were determined and are presented below: 10 20 30 38 Chain A: SYG(C)YCGAGGGG(W)PQDASDRCCFZHBCCYAKLTGCBPT 10 20 30 34 Chain B: pERDTKZBZVCDPCGCEIMGCDTAAAICFEDSEDG 10 14 Chain C: pEFSPEBCZGESZPC Papers presented by Aird 12 - 1 at the 8th World Congress on Animal, Plant and Microbial Toxins and published in Biochemistry have presented the three crotapotin chains, chains C and A being complete and chain B incomplete. Inter-Cro The same methodology 14 used for crotoxin separation, was used for the Inter- Cro separation except that the dimension of the column used was three times that of the previous system for the same venom load. A fraction between crotox¬ in and crotamine, with the same characteristic as crotoxin was thus obtained. When this fraction was submitted to the same procedure as used to obtain crotapo¬ tin, via SP-Sephadex, it unfolded into the two fractions mentioned earlier (crotapo¬ tin and phospholipase A 2 ). Studies currently underway will confirm whether this is an iso-crotoxin. Different isoforms of crotoxin have been mentioned by Faure and Bon 6 ' 7 ' 4 . In our Laboratory we observed that Inter-Cro is much more insoluble than crotoxin, and has when injected in mice intraperitonially, a delayed toxic effect i.e. 20-30 minutes compared to 3-5 min. with crotoxin. í, i SciELO 18 Mem. Inst. Butantan, 52 (supl.), 1990 REFERENCES 1. AIRD, S.D.; KAISER, I.I.; LEWIS, R.V.; KRUGGEL, G.W. Rattlesnake presynaptic neu- rotoxins; primary structure and evolutionary origin of the acidic subunit. Biochemistry, 24:7054-7058, 1985. 2. BON, C. & JENG, T.W. In: CECCARELLI & CLEMENT, ed. Advances in cytopharma- cology. New York, Raven Press, 1979. 3. BON, C.; CHANGEUX, J.P.; JENG, T.W.; FRAENKEL-CONRAT, H. Eur. J. Biochem., 99: 471-481, 1979. 4. BON, C.; CHOUMET, V.; FAURE, G.; YIANG, M.S.; LEMBEZAT, M.P.; RADVANYI, F.; SALIOU, B. Crotoxin, half-century of investigations on a phospholipase neurotoxin. In: SIMPÓSIO ANUAL DA ACIESP SOBRE TOXINAS PROTÉICAS, 12. Anais. Cam¬ pinas, ACIESP, 1988. p. 9-18. 5. BREITHAUPT, H.; RUBSAMEN, K.; HABERMAN, E. Eur. J. Biochem., 49:333, 1974. 6. FAURE, G. & BON, C. Several isoforms of crotoxin are present in individual venoms from the South American rattlesnake Crotalus durissus terrificus. Toxicon, 25: 229-234, 1987. 7. F^URE, G. & BON, C. Crotoxin, a phospholipase A 2 neurotoxin from the South Ameri¬ can rattlesnake Crotalus durissus terrificus', purification of several isoforms and com- parison of their molecular structure and of their biological activities. Biochemistry, 27: 730-738, 1988. 8. FRAENKEL-CONRAT, H; JENG, T.W.; HSTANG, M. Biological activities and amino acid sequence of crotoxin B. In: FAKER, D & WADSTRÕM, T. ed. Natural toxins. Intern-Symps. Animal, Plant and Mícrobial Toxins, 6. Proc. Oxford, N.Y., Perga- mon Press, 1980. 9. HENDON, R.A. & FRAENKEL-CONRAT, H. Proc. Natl. Acad. Sei. USA, 68: 1560, 1971. 10. HORST, J.; HENDON, R.A.; FRAENKEL-CONRAT, H. Biochem. Biophys. Res. Comm., 46: 1042, 1972. 11. JENG, T.W.; HENDON, R.A., FRAENKEL-CONRAT, H.L. Proc. Natl. Acad. Sei. USA, 75: 600, 1978. 12. KAISER, I.I.; AIRD, S.D.; KRUGGEL, W.; LEWIS, R.V. In: WORLD CONGRESS ON ANIMAL, PLANT AND MÍCROBIAL TOXINS, 8. United Kingdom, Newcastle upon Tyne, 1985. p. 579. 13. LAURE, C.J. (1980). MastePs Thesis, Gabilan N. (1984). Estrutura primária da cadeia C da crotapotina, FMRP-USP; Conti, M.A.B. (1986). Estrutura primária da cadeia B da crotapotina. Doctoral Thesis, FMRP-USP and Estrutura primária da cadeia A, Saito, Y. e Laure, C.J. FMRP-USP (1986). 14. LAURE, C.J. Oral communication. Simpósio Anual da ACIESP, 12. Anais. 1988. 15. RÜBSAMEN, K; BREITHAUPT, H.; HABERMAN, E. Naunyn-Schmie. Arch. Exp. Path. Pharmak., 279: 274, 1971. 16. SLOTTA, K. & FRANKEL-CONRAT, H. Ber. Dtsch. Chem. Ges., 71: 1076-1081, 1938. 17. VITAL BRAZIL, 0. Pharmacology of crystalline crotoxin. II. Neuromuscular blocking action. Mem. Inst. Butantan, 33: 981-992, 1966. 19 JSciELO Mem. Inst. Butantán, 52 (supl.), 1990 CROTOXIN, THE PHOSPHOLIPASE A 2 NEUROTOXIN FROM THE VENOM OF CROTALUS DURISSUS TERRIFICUS Barbara J. Hawgood, Division of Biomedical Sciences, King's College London, Kensington, Lond, W8 7AH, England. Death following systemic crotoxin poisoning of experimental animais is due to respiratory paralysis of peripheral origin. 8 The primary defect lies in a failure of nerve impulses to release acetylcholine following arrival at motor nerve termi¬ nais but the molecular events are ill-understood. Crotoxin consists of two poly- peptide subunits, a weakly toxic, basic phospholipase A 2 and a non-toxic acidic component devoid of enzymatic activity. Evidence suggests that the phospholi¬ pase A 2 subunit binds to specific sites at the motor nerve terminal and that the role of the acidic component is to reduce non-specific binding by chaperoning the phospholipase A 2 subunit to these sites. 1 Unlike botulinum toxin, crotoxin does not cross the axolemma by receptor-mediated endocytosis to act in- ternally. 7 Electrophysiological recordings from single endplates in frog and mouse nerve- muscle preparations show that crotoxin induces a series of changes in the amount of acetylcholine release per impulse viz. an initial fali, a secondary rise and a ter- tiary fali leading to complete inhibition of evoked release. The rate of spontane- ous transmitter release initially falis in parallel but at intoxicated frog endplates, the secondary rise is slower and far more sustained. 4 ' 5 The secondary increase in evoked transmitter release from intoxicated mouse motor nerve terminais is due, at least in part, to toxin blockade of a class of K + channels and so to a greater Ca 2+ influx. 6 However, such a blockade has not been observed in the frog and another mechanism must be operative 6 as is also suggested by the in¬ crease in facilitation of endplate potential (e.p.p.) amplitude due to closely spaced twin impulses. 5 In the tertiary phase when transmitter release is declining towards zero, twin-impulse facilitation is reduced, delay time between stimulus and onset of the e.p.p. is increased and the rise-time of the e.p.p. is slowed. 5 Phospholipase A 2 activity has been implicated in the secondary as well as ter¬ tiary phases of intoxication in the frog. 4 However, there is no evidence that prostaglandin synthesis is involved in any of the stages as pretreatment with inhi- 21 cm 2 3 z 5 6 11 12 13 14 15 16 Mem. Inst. Butantan, 52 ( supl .), 1990 bitors of arachidonate metabolism such as aspirin and nordihydroguaiaretic acid failed to alter the characteristic changes in e.p.p. amplitude and miniature e.p.p. frequency induced by crotoxin. 2 Similarly there is no evidence that crotoxin in- itiates the activation of protein kinase C as pre-treatment with the blocker H-7 did not change the characteristic pattern of intoxication. 5 It has been proposed that crotoxin's neurotoxicity is due to site-directed hydrol- ysis of the axolemma 4 affecting the release process per se. 5 This leads to a ser¬ ies of time-gated changes which include transient facilitation then uncoupling of phasic release and generalized acceleration of spontaneous release. Progressive hydrolysis leads to necrosis of the nerve terminal. Crotoxin binds to other sites less readily and focal lesions in the kidney, 3 skeletal muscle and lungs 4 have been reported following intoxication of experimental animais. REFERENCES 1. BON, C., BOUCHIER, A., CHOUMET, V.; FAURE, G.; JIANG, M.S. LAMBEZAT, M.R.; RADVANYI, F.; SALIOU, B. Crotoxin, half-century of investigations on a phospholi- pase A 2 neurotoxin. Acta Physiol. Pharmacol. Latinoam., 39: 439-448, 1989. 2. EDWARDS, J.; FIAWGOOD, B. J; SMITPI, I.C.H. The effects of inhibitors of arachidonate metabolism on spontaneous and evoked endplate potentials at normal and crotoxin- treated frog neuromuscular junctions. Toxicon, 28: 985-988, 1990. 3. HADLER, W.A. & VITAL BRAZIL, 0. Pharmacology of crystalline crotoxin. IV. Nephrotox- icity. Mem. Inst. Butantan, 33: 1001-1008, 1966. 4. FIAWGOOD (Exceli), B.J. Plow similar are the actions of crotoxin and /3-bungarotoxín? Acta Physiol. Pharmacol. Latinoam., 39: 397-406, 1989. 5. RODRIGUES-SIMIONI, L.; FIAWGOOD, B.J.; SMITH, I.C.H. Properties of the early phases of crotoxin poisoning at frog neuromuscular junctions. Toxicon in press. 6. ROWAN, E.G. & FIARVEY, A.L. Potassium channel blocking actions of /3-bungarotoxin and related toxins on mouse and frog motor nerve terminais. Brit. J. Pharmacol., 94: 839-847, 1988. 7. TRIVEDI, S.; KAISER, I.; TANAKA, M.; SIMPSON, L. Pharmacologic experiments on the interaction between crotoxin and the mammalian neuromuscular junction. J. Pharmacol. exp. Ther., 25 7:490-496, 1989. 8. VITAL BRAZIL, O. Venoms: Their inhibitory action on neuromuscular transmission. Int. Encycl. Pharmacol. Ther., 74:145-167, 1972. 22 I, | SciELO Mem. Inst. Butantan, 52 (supl.l, 1990 PHARMACOLOGY OF CROTAMINE Oswaldo Vital Brazil, Department of Pharmacology, Faculty of Medicai Sciences, State University of Campinas, Campinas, São Paulo. Crotamine is a basic polypeptide toxin present in the venom of a variety of Crotalus durissus terrificus ( C.d. terrificus var. crotaminicus). The LD50 of this toxin i.v. in mice and its 95% confidence intervals are 1,500 (1,010-2,230) /xg/kg. Crotamine is considerably (about 18 times) less toxic than crotoxin (LD50 82 /tg/kg), the main toxin from the South American rattlesnake venom. Crotamine activates the sodium channel exclusively or mainly in the skeletal muscle fibre membrane. All effects produced by it are assigned to this action. They are in iso- lated or in situ neuromuscular preparations of mammals: 6 ' 4 i. fibrillation and/or a sustained contraction of short duration usually only observed immediately after the injection or addition of the toxin to the bath; ii. a delay of relaxation of the muscle and/or an aftercontraction following the muscle response to direct or in- direct stimulation with single or high frequency shocks; iii. an increase of the twitch tension in muscles stimulated directly or indirectly with maximal shocks. These effects, also evoked in curarized muscle are accompanied either (effects referred to in i. and ii.) by discharges of potentials of high frequencies (over 200-300 per sec.) and small amplitude (50-150 /xV), or (effect referred to in iii,) by more than one action potential. Ca + + , Mg + 4 and quinine or quinidine antagonize these ef¬ fects. Crotamine may also depress the responses to direct or indirect stimula¬ tion, and in large doses, to render the muscles inexcitable. Dilatation of the sarcoplasmic reticulum and myonecrosis, effects evoked by all toxins that acti- vate the sodium channel, are induced by crotamine. 1 They are explained by an increase in Na + influx leading to increased water influx, cell swelling, and even- tually cell death 3 . The action of crotamine in the sodium channel was demonstrated in the rat diaphragm. 4 ' 5 The depolarization produced by crotamine in curarized or noncura- rized muscles was prevented by tetrodotoxin or low sodium in the bathing fluid. Crotamine produces in nonanesthetized animais, especially in goats, myotonia- like symptoms. 4 ' 6 Myotonia may be caused either by an increase in muscle fibre membrane resistance due to a decrease in its conductance to Cl" or to an in¬ crease in the membrane conductance to Na + . The selectivity of action of crota¬ mine in producing activation of the sodium channel in skeletal muscles is responsible for inducing myotonia-like effects in animais. 23 cm 2 3 z 5 6 11 12 13 14 15 16 Mem. Inst. Butantan, 52 (supl.), 1990 The participation of crotamine in the genesis of the myonecrosis observed in C.d. terrificus snake-bite accidents 2 is doubtful owing to its low activity and con- centration in the venom. REFERENCES 1. CAMÉRON, D.L. & TU, A.T. Chemical and functional homology of myotoxin a from prairie rattlesnake venom and crotamine from South American rattlesnake venom. Biochem. Biophys. Acta., 532: 147-154, 1978. 2. CUPO, P.; AZEVEDO-MARQUES, M.M.; HERING, S.E. Clinicai and laboratory features of South American rattlesnake Crotaius durissus terrificus envenomation in children. Trans. Ftoyal Soc. trop. Med. Hyg., 82: 924-929, 1988. 3. OWNBY, C.L. Pathology of rattlesnake envenomation. In: TU, A.T. ed. Rattlesnake ve- noms; theiractions and treatment. New York, Marcei Dekker, Inc., 1982. p. 163-209. 4. PELLEGRINI FILHO, A. Contribuição ao estudo da ação da crotamina no músculo es¬ quelético. Tese de Doutoramento, Faculdade de Ciências Médicas, Universidade Estadual de Campinas, 1976. 5. PELLEGRINI FILHO, A.; VITAL BRAZIL, O.; FONTANA, M.D.; LAURE, C.J. The action of crotamine on skeletal muscle: an electrophysiological study. In.: ROSENBERG, P. ed. Toxin: Animal, Plant and Microbial. New York, Pergamon Press, 1978. p. 375-382. 6. VITAL BRAZIL, O. & PRADO-FRANCESCHI, J. Repetitive muscle responses induced by crotamine. Toxicon, 77:61-67, 1979. 24 ], | SciELO Mem. Inst. Butantan, 52 (sjupl.), 1990 ON THE PHARMACOLOGY OF CONVULXIN AND GYROXIN Júlia Prado-Franceschi, Department of Pharmacology, Faculty of Medicai Sciences, State University of Campinas, Campinas, São Paulo. The venom of the South American rattlesnake C.d. terrificus is known to con- tain several neurotoxins: crotoxin 10 , crotamine 5 , gyroxin 2 and convulxin 6 . Croto- xin is the main neurotoxin of this venom and is the component responsible for its very high toxicity 12 . Crotamine is a basic polypetide which, unlike crotoxin, is present only in the venom from certain regions 3 . However, the injection of crotoxin or of crotamine into experimental animais does not reproduce all the effects of C.d. terrificus- induced envenomation such as early respiratory and circulatory effects, convul- sions or the symptomatology resembling a labyrinthic lesion. This latter effect could be attributed to gyroxin 2 , a non-lethal neurotoxin. The most characteristic effect observed following injection of purified gyroxin consists of rolling movements in one direction for 5 to 20 minutes, after a lag time of some minutes. This syndrome gradually disappears and there is complete recovery. Purified gyroxin demonstrates TAME-esterase activity 1 . In 1967 we described a neurotoxic fraction responsible for the convulsions and early respiratory and circulatory disturbances 13 . From this fraction we isolated convulxin (Cx), a high molecular weight toxin which behaves as a homogeneous protein when examined by gel filtration chromatography and immunoelectrophoresis 6 ' 7 . When administered i.v. into conscious mice, Cx evokes within 20 seconds a brief phase of apnea, followed by loss of equilibrium, including tonic-clonic con¬ vulsions. In the cat, we also observed autonomic disturbances such as saliva- tion, flaccid paralysis, abdominal cramps and nistagmus. Intravenous administration of Cx into anesthetized dogs causes intense respira¬ tory stimulation followed by apnea of short duration as well as circulatory distur¬ bances characterized by an immediate and abrupt fali in blood pressure followed by a transient hypertension. 25 cm 2 3 L 5 6 11 12 13 14 15 16 Mem. Inst. Butantan, 52 (supl.), 1990 Cx produces a rapid thromb ocy topenía "in vivo” associated with respiratory and circulatory changes, suggesting that they are produced-by pharmacological- ly active substances which Cx liberates from platelets. In studying the Cx activated guinea-pig platelets we have verifíêd that this Cx concentration-dependent aggregation is followed by the release of ATP and by the formation of thromboxanes 11 , Cx-induced platelet aggregation was inhibit- ed by EDTA and by prostacyclin. As Cx also induces leukopenia 8 , the participa- tion of leukopytes or of any substance that they may produce, is not excluded. Cx is extremely feeble when administered by the i.m. or-s.c. route. This could be related to poor absorption due to its high molecular wei^ht. The significance of Cx in the symptoms caused by South American rattlesnake bite is still not clear. The high content of Cx in C.d. cascavella venom could be relevant. The appear- ance of convulsive movements in some victims 4 ' 9 strongly suggests that this tox- in may participate in envenomation. REFERENCES 1. BARRABIN, H.; MARTIARENA, J.L.; VIDAL, J.C.; BARRIO, A. Isolation and charac- terization of gyroxin from Crotalus durissus terrificus venom. In: ROSENBERG, P. ed. Toxins: Animal, plant and mlcrobial. Oxford, Pergamon Press, 1978. p. 113. 2. BARRIO, A. Gyroxin a new neurotoxin of Crotalus durissus terrificus venom. Acta. Physiol. Latinoam., 7 7(4):222, 1961. 3. BARRIO, A. & VITAL BRASIL, O. Neuromuscular action of the Crotalus durissus ter¬ rificus (Laur.) poisons. Acta Physiol. Latinoam., 7:291, 1951. 4. BRAZIL, V. La défense contre 1'ophidisme. 2.ed. São Paulo, Pocai and Weiss Ed., 1914. 5. GONÇALVES, J.M. & VIEIRA, L.G. Estudos sobre venenos de serpentes brasileiras. I. Análise eletroforética. Anais Acad. bras. Ciênc., 22: 141, 1950. 6. PRADO-FRANCESCHI, J. Estudo sobre convulxina, Tese de doutorado, UNICAMP, Campinas—SP, 1970. 7. PRADO-FRANCESCHI, J. & VITAL BRAZIL, O. Convulxin, a new toxin from the ven¬ om of the South American rattlesnake Crotalus durissus terrificus. Toxicon, 79:875, 1981. 8. PRADO-FRANCESCHI, J.; TAVARES, D.Q.; HERTEL, R.; LÔBO de ARAÚJO, A. Ef- fects of convulxin a toxin from rattlesnake venom, on rabbit platelets. Toxicon, 79:661, 1981. 9. SEBA, R. Personnal communication. 10. SLOTTA, C.H. & FRAENKEL-CONRAT, H. Estudos químicos sobre os venenos ofídi- cos. Purificação e cristalização do veneno de cobra cascavel. Mem. Inst. Butan¬ tan, 72:505, 1938-39. 11. VARGAFTIG, B.B.; PRADO-FRANCESCHI, J.; CHIGNARD, M.; LEFORT, J.; MARLAS, G. Activation of guinea-pig platelets induced by convulxin, a substance extracted from the venom of Crotalus durissus cascavella. Eur. J. Pharmacol., 68: 451, 1980. 12. VITAL BRAZIL, O.; PRADO-FRANCESCHI, J.; WAISBICH, E. Pharmacology of crys- talline crotoxin. I. Toxicíty. Mem. Inst. Butantan, 33: 973, 1966. 13. VITAL BRAZIL, O., PRADO-FRANCESCHI, J.; WAISBICH, E. Fator neurotóxico na peçonha de Crotalus durissus terrificus diferente da crotoxina e da crotamina. Ciênc. Cult. São Paulo, 79:658, 1967. í, i SciELO 26 Mem. Inst. Butantan, 52 (supl.), 1990 RATTLESNAKE BITES. CLINICAL FEATURES AND COMPLEMENTARY TESTS Marisa M. de Azevedo Marques' 1 , Patmira Cupo 1 , Carlos Fa¬ ria S. Amaral 2 , Sylvia E. Hering\ 1. Faculty of Medicine of Ribeirão Preto, University of São Paulo, and 2. Faculty of Medi¬ cine, Federal University of Minas Gerais. Bites by snakes of the genus Crotalus in Brazil are important npt only because of their high incidence in certain regions, but also because of their potential produc- tion of severe and even fatal clinicai pictures. Six subspecies have been identified which are distributed irregularly through- out the Brazilian territory, the occurrence of snakebites being predominant in the Southeast of the country. The subspecies most commonly encountered in the State of São Paulo is C. durissus terrificus. The frequency of bites by the South American rattlesnake is lower than that of bites by snakes of the genus Bothrops. Data provided by the Butantan Insti- tute (Vital Brazil Hospital, 1966-1977) and the Yearly Statistical Data published by the Health Ministry in 1989 reveal that 8 to 10% of poisonous snakebites are caused by rattlesnakes. In the Ribeirão Preto region (data provided by the Center of Intoxication Control) and in the Botucatu region 6 , this percentage reaches 20-25%. Until the beginning of the 1980's, the South American rattlesnake venom was reported to have major activities of the hemolytic and neurotoxic type, the most serious complication of human envenomation being acute tubular necrosis, whose pathogeny was interpreted to be due to the hemolytic effect of the venom as- sociated or not with a direct nephrotoxic effect 15 . Studies initiated in 1982 on victims of rattlesnake envenomation using clinicai observation, laboratory tests and biopsy of the member contralateral to the bit- ten one demonstrated the systemic myotoxic action of C. durissus terrificus ven¬ om, characterized by aggression to skeletal muscle fibers 4,5,7,12,13,16 More recently, the analysis of serum leveis of the isoenzymes creatine kinase (CK) and 27 P 'SciELO 0 11 12 13 14 15 16 1 Mem. Inst. Butantan, 52 (supl.), 1990 lactate dehydrogenase (LDH), together with the focal involvement of skeletal mus- cle detected in the biopsies of these patients, has led to the conclusion that the venom may be selective for oxidative type I and/or type lia fibers 8 . This hypothe- sis is supported by histochemical data obtained in the analysis of biopsies from two patients, which demonstrated selective atrophy of type I fibers 9 . In 1986, the existence of an enzyme with thrombin-like activity was identified in snake venom. This enzyme has a clotting effect on human plasma in vitro and may elicit fibrinogen consumption 14 , with reports of afibrinogenemia and ab- sence of blood clotting without platelet consumption 2 being available. In 1987, the idea of a hemolytic activity of the South American rattlesnake venom in humans was discarded through specific laboratory tests such as serum hemoglobin and haptoglobin measurements and absence of hemoglobin in urine 5 . On the basis of these data, the venom of C. durissus terrificus has been reported to have major activities of the neurotoxic and systemic myotdxic type, in addition to a thrombin-like clotting action, to which the main clinicai manifestations de¬ tected in human poisoning have been attributed. Clinicai signs and symptoms attributed to the neurotoxic activity of the ven¬ om: usually evident during the first hours, they are characterized by a myasthe- nic fácies (the "neurotoxic” fácies of Rosenfeld 15 ), anisocoria, uni- or bilateral palpebral ptosis, ophthalmoplegia with possible difficulty in accommodation and diplopia. Less frequent manifestations are velopalatine paralysis with difficulty in swallowing and decreased vomiting reflex, with taste and smell alterations. Clinicai signs and symptoms attributed to the myotoxic activity of the venom: generalized muscle pains may appear early. The reddish or darker (as dark as brown) color of urine is a symptom of later onset reflecting the elimination of my- oglobin, a pigment released from muscle tissue, which represents the most evi¬ dent manifestation of rhabdomyolysis. Clinicai signs and symptoms attributed to the clotting activity of the venom: lack of blood clotting or an increase in clotting time may occur in 30 to 50% of patients, with the occurrence of slight bleeding usually limited to the gingiva 10 Acute respiratory failure, seldom reported for human snakebite victims, has been observed between the 3rd and 1 2th day after the bite in patients who de- velop simultaneous acute renal failure. More recent reports have described pa¬ tients with respiratory symptoms occurring during the first 24 hours after the bite in the absence of renal failure and lasting no longer than one day 1 ■ 11 -i 2 . Impair- ment of respiratory muscles as a consequence of the neurotoxic and myotoxic activities of the venom has been pointed out as the mechanism of the ventilatory disorders occasionally observed in victims of rattlesnake bites 16 ' 17 . Similarly, limb paralysis and fasciculations and tremors are clinicai manifesta¬ tions of low frequency which are interpreted as being due to the neurotoxic and/or myotoxic activity of the venom. Since the venom is devoid of "proteolytic" activity, only the marks of the fangs are observed at the site of the bite, with discrete or absent edema and/or erythema. Laboratory Diagnosis As a result of myolysis, enzymes are released into the blood stream, with elevat- ed CK and LDH leveis, as welí as aspartate aminotransferase (AST) and aldolase leveis. The increase in CK leveis occurs early, reaching a peak within the first 24 hours after the bite, whereas LDH leveis increase in a slower and more gradual manner. 28 i, i SciELO Mem. Inst. Butantan, 52 (supl.), 1990 Analysis of CK and LDH isoenzymes reveals an infarct-like pattern, with an in- crease in the so-called cardiac fractions CK-MB and LD,. Electrocardiogram and serial two-dimensional ecocardiogram studies carried out on 4 patients with se- vere poisoning ruled out the possibility of aggression to the myocardium 8 . Nonspecific blood count changes occurs, with possible leucocytosis with neu- trophilia and a leftward shift, at times with the presence of toxic granulations. An increase in clotting time, prothrombin time and partially activated throm- boplastin time may occur, as well as a decrease in fibrinogen. The urinary sediment is usually normal in the absence of renal failure, with the possible presence of discrete proteinuria not accompanied by hematúria. The presence of myoglobin in urine can be detected by the benzidine test or using urinalysis strips (a positive reaction also for hemoglobin) or by more specif- ic immunochemical methods such as immunoelectrophoresis, immunodiffusion tests and latex myoglobin agglutination test. When oligúria or anuria sets in, elevated urea, creatinine, uric acid, phospho- rus and potassium leveis are observed, as well as decreased calcium leveis. The muscle biopsy findings obtained from patients bitten by C. durissus terrifi¬ cus reveal a wide spectrum of ultrastructural alterations involving muscle fibers, such as focal points of myonecrosis with myofibril disintegration, myofilament disorganization and lysis, loss of transversal striations, dilatation of the sarcoplas- mic reticulum, formation of contraction bands and mytochondrial edema, side by side with intact fibers in the same muscle T 5 ,7,12,13,16 The clinicai and laboratory data described above refer to envenomation caused by C. durissus terrificus and C. durissus collilineatus. Recently, Fan et al. 18 , reported on two snakebite victims from an area in which C. durissus cascavella is prevalent, whose clinicai and hematological data were similar to those previ- ously reported in the literature. REFERENCES 1. ALVARES, J.M. Tratamento intensivo da paralisia respiratória no acidente crotálico. Belo Florizonte, 1980. 54p. (Tese de mestrado — Faculdade de Medicina da UFMG). 2. AMARAL, C.F.S.; REZENDE, N.A.; PEDROSA, T.M.G., Da SILVA, O.A.; PEDROSO, E.R.P. Afibrinogenemia secundária a acidente ofídico crotálico (Crotalus durissus terrificus). Rev. Inst. Med. Trop. S. Paulo, 30: 288-292, 1988. 3. AMORIM, M. de F. & MELLO, R.F. Nefrose do nefron intermediário no envenenamento crotálico humano. Estudo anátomo-patológico. Mem. Inst. Butantan, 24: 281-316, 1952. 4. AZEVEDO-MARQUES, M.M.; CUPO, P., COIMBRA, T.M., HERING, S.E., ROSSI, M.A.; LAURE, C.J. Myonecrosis, myoglobinuria and acute renal failure induced by South American rattlesnake (C. durissus terrificus) envenomation in Brazil. Toxicon, 213: 631-636, 1985. 5. AZEVEDO-MARQUES, M.M.; HERING, S.E. CUPO, P. Evidence that Crotalus durissus terrificus (South American rattlesnake) envenomation in humans causes myolysis rather than hemolysis. Toxicon, 11: 1163-1168, 1987. 6. BARRAVIEIRA, B. Curso sobre acidentes por animais peçonhentos; acidentes por ser¬ pentes do gênero Crotalus. Arq. bras. Med., 64: 14-20, 1990. 7. CUPO, P., AZEVEDO-MARQUES, M.M.; HERING, S.E. Clinicai and laboratory features of South American rattlesnake IC. durissus terrificus) envenomation in children. Trans. Royal Soc. trop. Med. Hyg., 82: 924-929, 1988. 8. CUPO, P.; AZEVEDO MARQUES, M.M.; HERING, S.E. Acute myocardial infarction-like enzyme profile in human victims of C. durissus terrificus envenoming. Trans. Royal Soc. trop. Med. Hyg., 84: 447-451, 1990. 29 cm 2 3 L 5 6 11 12 13 14 15 16 Mem. Inst. Butantan, 52 (supl.), 1990 9. JORGE, M.T.; PEREIRA, J.R.; BONFATI JR., W.; RIBEIRO, L.A.; AMATO NETO, V,; RASSI, I.E.; BOUABCI, A.S Comportamento de enzimas e alterações anátomo- patológicas do músculo esquelético em pacientes picados por serpentes do gênero Crotalus. Rev. Soc. bras. Med. Trop., 19 (suppl.): 27, 1986. 10. KAMIGUTI, A.S. & CARDOSO, J.L.C. Haemostatic changes caused by the venoms of South American snakes. Toxicon, 27: 955-963, 1989. 11. LOPEZ, M.; FOSCARINI, L; ALVARES, J.M.; DINIZ FILHO, L; MARSA, V.D.; PROCÓ- PIO, N.P.M. Tratamento intensivo das complicações do acidente ofídico. Rev. Ass. med. Minas Gerais, 23: 107-112, 1972. 12. MAGALHÃES, R.A. Contribuição ao estudo da rabdomiólise no acidente ofídico crotáli- co. Belo Horizonte, 1986. 90p. (Tese de mestrado — Faculdade de Medicina da UFMG). 13. MAGALHÃES, R.A.; RIBEIRO, M.M.F.; REZENDE, N.A.; AMARAL, C.F.S. Rabdomió¬ lise secundária a acidente ofídico crotálico (C. durissus terrificus). Rev. Inst. Med. trop. S. Paulo, 28: 228-233, 1986. 14. RAW., I.; ROCHA, M.C.; ESTEVES, M.I.; KAMIGUTI, A.S. Isolation and characteriza- tion of a thrombin-like enzyme from the venom of Crotalus durissus terrificus. Braz. J. Med. Blol. Res., 19: 333-338, 1986. 15. ROSENFELD, G. Symptomatology, pathology and treatment of snakebites in South America. In: BUCHERL, W.; BUCKLEY, E.E.; DEULOFEU, V. ed. Venomous animais and their venoms. New York, Academic Press, 1971. p. 345-384. 16. ROSSI, M.A.; PERES, L.C.; DEPAOLA, F.; CUPO, P.; HERING, S.E; AZEVEDO- MARQUES, M.M. Electron microscopic study of systemic myonecrosis due to poison- ing by tropical rattlesnake IC. durissus terrificus) in humans. Arch. Path. Lab. Med., 113: 169-173, 1989. 17. VITAL BRAZIL, O. Venenos ofídicos neurotóxicos. Rev. Ass. Med. Bras., 26: 212-218, 1980. 18. FAN, H.W.; OLIVEIRA, J.R.F.; MEDEIROS, I.M.; PINHEIRO, E.M.; FRANÇA, F.O.S.; CASTRO, S.; KAMIGUTI, A.S.; CARDOSO, J.L.C. Aspectos regionais do en¬ venenamento crotálico: acidente em área de Crotalus durissus cascavella: In CON¬ GRESSO DA SOCIEDADE BRASILEIRA DE MEDICINA TROPICAL, 26. Resumo 70, 1990. p.94 30 cm SciELO 10 11 12 13 14 15 16 Mem. Inst. Butantan, 52 (supl.), 1990 PHARMACOLOGY OF CORAL SNAKE VENOMS Oswaldo Vital Brazil, Department of Pharmacology, Faculty of Medicai Sciences. State University of Campinas, Campinas, São Paulo. Coral snakes, the New World Elapidae are included in the genera Micruroides and Micrurus. The genus Micrurus comprises nearly all human snake-bite acci- dents. Coral snake venoms as the venoms of other Elapidae are neurotoxic, producing loss of muscle strength and, in general, death by respiratory paralysis of peripheral origin in animais and humans. They display phospholipase A 2 ac- tivity. Proteolysis activity is absent or of very low grade. The main toxins from elapid venoms are: 1. postsynaptic neurotoxins; 2. presynaptic neurotoxins; 3. cardiotoxins; and 4. myotoxic phospholipase A 2 . From the study of the mechanisms of action of the coral snake venoms so far carried out, it can be deduced that postsynaptic neurotoxins are probably com- ponents of all coral snake venoms while presynaptic neurotoxins and cardiotox¬ ins or myotoxic phospholipases occur in some of them. We can, therefore, divide the coral snake venoms according to their mode of action in 5 : 1. coral snake ven¬ oms that exert only postsynaptic neurotoxin-like action (blockade of end-plate receptors); 2. coral snake venoms that block the end-plate receptors (postsynaptic neurotoxin-like action) and inhibit evoked acetylcholine release by the motor nerve endings (presynaptic neurotoxin-like action); 3. coral snake venoms that block the end-plate receptors (postsynaptic neurotoxin-like action) and depolarize the muscle fibre membrane (cardiotoxin or myotoxic phospholipase A 2 -like action). Belong to group 1 by their mode of action the venoms of M. frontalis 7 and M. lemniscatusf 1 . The first species is distributed over centraleastern, central- western and Southern Brazil and is also found in Argentine, Uruguay and Paraguay. M. lemniscatus occurs in the Guyanas, Trinidad, Venezuela, Colombia, Equador, Bolivia and in northern, northeastern and central Brazil until Paraná and Mato Grosso. M. corallinus venom 6 blocks the end-plate receptor and the evoked acetylcho¬ line release by the motor nerve endings (group 2 by its mode of action). M. coral¬ linus is distributed over central and Southern Brazil, south of Amazon Basin and northern Argentine. The venoms of M. fulvius 3 ' 8 and M. nigrocinctus T 2 exert postsynaptic neurotoxin-like action and depolarization of the muscle fibre membrane. M. ful¬ vius is a coral snake species from Southern United States and northeastern Méxi¬ co. M. nigrocinctus occur from South of México to North of Colombia. 31 cm 2 3 L 5 6 11 12 13 14 15 16 Mem, Inst. Butantan, 52 (supl.), 1990 REFERENCES 1. DALÔ, N. eta/. Neuromuscular blocking activity of a fraction isolated from the venom of cõral snake, Micrurus nigrocinctus. In: SYMP. ON ANIMAL, PLANT AND MICROBI- AL TOXINS. Abstract of Papers, 1986. 2. RODRIGUES-SIMIONI, L. Personal communication, 1990. 3. SNYDER, G.K.; RAMSEY, H.W.; TAYLOR, W.J.; CHIOU, C.Y. Neuromuscular block- ade of chick biventer cervicis nerve muscle preparation by a fraction from coral snake venom. Toxicon, 7 7:505-508, 1973. 4. VITAL BRAZIL, O. Ação neuromuscular da peçonha de Micrurus. São Paulo, 1963. (Tese de doutoramento-Faculdade de Medicina — USP). 5. VITAL BRAZIL, O. Coral snake venoms; mode of action and pathophysiology of ex¬ perimental envenomation. Rev. Inst. Med. trop. São Paulo, 29: 119-126, 1987. 6. VITAL BRAZIL, O. & FONTANA, M.D. Ações pré-juncionaís e pós-juncionais da peço¬ nha de cobra coral Micrurus corallinus na junção neuromuscular. Mem. Inst. Butan¬ tan, 47/48:13-26, 1983/84. 7. VITAL BRAZIL, O.; FONTANA, M.D., PELLEGRINI, A. Physiopathologie et thérapeu- tique de 1'envenomation expérimentale causées par le venin de Micrurus frontalis. Mem. Inst. Butantan, 40/4 7:221-240, 1976/77. 8. WEISS, R. & McISAAC, R.J. Cardiovascular and muscle effects of venom from coral snake venom, Micrurus fulvius. Toxicon, 9: 219-228, 1971. 32 I, | SciELO Mem. Inst. Butantan, 52 (supl.), 1990 ELAPIDIC ENVENOMATION: CLINICAL FEATURES Fábio Bucaretchi, Centro de Controle de Intoxicações, Facul¬ dade de Ciências Médicas, Universidade Estadual de Campi¬ nas, Campinas, São Paulo. The coral snakes, which belong to the Micrurus genus and Elapidae family, rarely determine accidents in human beings in Brazil. According to data of the Health Ministry, of 20,884 notified snakes accidents, in the whole country, bet- ween June 1986 and December 1 987, only 0.7% (141) of the accidents were determined by snakes of this genus, and the lethality coeficient observed was 0 . 7 % 1 Coral snake venom is neurotoxic, acting on the neuromuscular junction. It de¬ termines a myasthenia gravis -like syndrome, of variable intensity 3 ' 4 . Initiaily, the patient may develop vomiting and mild local pain, usually followed by local pa- resthesia and myalgia with proximal progression tendency. Posteriorly, Progres¬ sive muscular weakness may develop, with palpebral ptosis, externai opthtalmoplegia, respiratory difficulties, disphagia, mandibular ptosis, flaccid pa- ralysis affecting the neck, trunk and limbs. 2 ' 4 ' 5 ' 6 In these accidents, in addition to antivenom and adequate ventilatory suport, when determined by M. frontalis, whose neurotoxin competes with acetylcholi- ne at the end-plate receptors of the neuromuscular junction, pharmacologic treat- ment with anticholinesterase drugs is possible. 2 ' 3 ' 4 ' 5 ' 6 REFERENCES 1. MINISTÉRIO DA SAÚDE. Secretaria Nacional de Ações Básicas da Saúde. Análise epi- demiológica dos acidentes ofídicos. Brasil, junho de 1986 a dezembro de 1987. Brasília, 1989. 2. VIEIRA, R.J.; BUCARETCHI, F.; ZAMBRONE, F.A.D. Acidente elapídico: relato de 7 acidentes e apresentação de "bloqueio farmacológico" com prostigmine em um caso. In: CONGRESSO BRASILEIRO DE TOXICOLOGIA, 6. Anais. São Paulo, 1987. Tema Livre 5.24. 33 cm 2 3 z 5 6 11 12 13 14 15 16 Mem. Inst. Butantan, 52 (supl.l, 1990 VITAL-BRAZIL, O. Venenos ofídicos neurotóxicos. Rev. Ass. med. bras., 26: 212-218, 1980. WARREL, A.D. Animal poisons. In: MANSON-BAHR, P.E.C. & BELL, D.R. ed. Man- son's tropical diseases. 19. ed. London, Bailiére Tindau, 1987. p. 855-898. WATT, G.; THEAKSTON, R.D.G.; HAYES, C.G.; YAMBAO, M.L.; SANGALANG, R.; RANOA, C.P.; ALQUIZALAS, E.; WARRELL, D.A. Positive response to edrophonium in patients with neurotoxic envenoming by cobras (Naja naja philippinensis). A pla¬ cebo controlled study. New Engl. J. Med., 315: 1444-48, 1986. ZANIN, M. & COELHO, L. Uso de neostigmine em acidente envolvendo Micrurus: des¬ crição de um caso. In: CONGRESSO BRASILEIRO DE MEDICINA TROPICAL, 26. Anais. Natal, 1990. Tema Livre 075. 34 cm SciELO 10 11 12 13 14 15 16 Mem. Inst. Butantan, 52 (supl.), 1990 SNAKE VENOM HEMORRHAGINS Fajga R. Mandelbaum, Serviço de Bioquímica, Instituto Butan¬ tan. São Paulo, SP. Due to the complexity of most snake venoms, different biological effects may be observed after envenomation. Bleeding is common in crotalinae envenoma- tion and is likely to result from one or more factors in the venom. The main fac- tors are hemorrhagic toxins and the others are components which produce a non clotting effect. The hemorrhagic principies destroy the basement membrane of the capillary vessels. However, damage of the endothelial cells can not be ruled out. These causes affecting the stability of the vessel wall allow the red blood cells to escape from the capillaries. A great number of metalloproteases have been isolated from different snake venoms and many of them were characterized as hemorrhagins. It is noteworthy that both hemorrhagic and nonhemorrhagic me¬ talloproteases of snake venoms can be similar in proteolytic specificity. Some ti¬ mes two metalloproteases isolated from the same venom can hydrolyze the same substrates, but one is hemorrhagic and the other is nonhemorrhagic or this acti- vity is only observed in very high doses compared to the hemorrhagic one. For instance the hemorrhagic factor HF 2 and bothropasin isolated from the venom of B. jararaca hydrolyze casein, B-chain of insulin, fibrinogen, and have similar molecular weights 49,100 and 48,000, respectively. However, HF 2 is 50 times more hemorrhagic than bothropasin. The hemorrhagins degrade fibrinogen, ac- ting mostly on A and B fí chain like some other snake metalloproteases. The clea- vage of insulin B-chain by venom hemorrhagic proteases is very similar to the cleavage of other venom metalloproteases nonhemorrhagic. Both show a prefe- rence to cleave on N-terminal side of Leu bonds. However, all snake venom he¬ morrhagins are distinguished from the other metalloproteases by their limited substrate specificity: the action on basement membrane of the capillary vessels. It was demonstrated with hemorragic toxins from C. afroxthat they degrade the components of the extracellular matrix of the capillary vessels, such as type IV collagen, laminin, and nidogen but not fibronectin 2 . Snake venoms from Asia and North America have been exhaustively studied and many hemorrhagic principies were isolated from them. As for the Latin Ame- 35 P 'SciELO 0 11 12 13 14 15 16 1 Mem. Inst. Butantan, 52 (supl.), 1990 rican snakes, hemorrhagins were isolated only from some Bothrops species and from Lachesis muta muta. The hemorrhagins isolated from venoms of Bothrops genus are structuraly similar proteins. They behave very similarly to the homolo- gous antigens on immunoprecipitation, C' fixation and neutralization reactions 1 ' 3 lt was also found that the Bothrops hemorrhagins have epitopes similar to the hemorrhagins of North American Crotalus and to the Asian Trimeresurus and Ag- kistrodon species. The Bothrops hemorrhagins have also few epitopes in com- mon with venoms of Vipera snakes. Although the antisera to the Bothrops hemorrhagic proteins do not cross react with these venoms, they neutralize par- tially their hemorrhagic activity, possible by steric hindrance of the active hemorr¬ hagic center or by some allosteric modification due to the combination of the immunoglobulins with the corresponding epitope(s). 4 Hemorrhagins of same molecular weight isolated from venoms of different sna- ke genus show a great structural homology. 5 On the other hand it was found that hemorrhagic and nonhemorrhagic metalloproteases isolated from the same sna- ke venom have about 75% sequence homology. 6 ' 7 The difference of the hemorr¬ hagic and nonhemorrhagic protein is located at the middle position 51-130 of the molecule. However, the determining residues positions that are responsible for the binding with the basement membrane to induce hemorrhage were not yet determined. REFERENCES 1. ASSAKURA, M.T.; REICHL, A.P.; MANDELBAUM, F.R. Comparison of immunologi- cal, biochemical and biophysical properties of three hemorrhagic factors isolated from the venom of Bothrops jararaca (jararaca). Toxicon, 24: 943-946, 1986. 2. BARAMOVA, E.N.; SHANNON, J.D.; BJARNASON, J.B.; FOX, J.W. Degradation of extracellular matrix proteins by hemorrhagic metalloproteinases. Arch. Biochem. Biophys., 275: 63-71, 1989. 3. MANDELBAUM, F.R. & ASSAKURA, M.T. Antigenic relationship of hemorrhagic fac¬ tors and proteases isolated from the venoms of three species of Bothrops snakes. Toxicon, 26: 379-385, 1988. 4. MANDELBAUM, F.R.; SERRANO, S.M.T.; SAKURADA, J.K.; RANGEL, H.A.; ASSA¬ KURA, M.T. Immunological comparison of hemorrhagic principies present in venom of the Crotalinae and Viperinae subfamilies. Toxicon, 27:169-177, 1988. 5. MIYATA, T.; TAKEY, H.; OZEY, Y.; ARAKAWA, M.; IWANAGA, S.; OMORI-SATOH, T. Primary structure of hemorrhagic protein, HR2a, isolated from the venom of Tri¬ meresurus flavoviridis. J. Biochem., 105, 847-853, 1988. 6. SHANNON, J.D.; BARAMOVA, E.N.; BJARNASON, J.B.; FOX, J.W. Amino acid se¬ quence of a Crotalus atrox venom metalloproteinase which cleaves Type IV colla- gen and gelatin. J. Biol. Chem., 264:1 1575-11583, 1989. 7. TAKUJA, H.; ARAKAWA, M.; MIYATA, T.; IWANAGA, S.; OMORI-SATOH, T. Primary structure of H 2 proteinase, a non-hemorrhagic metalloproteinase isolated from the venom of the Habu snake, Trimeresurus flavoviridis. J. Biochem., 106: 151-157, 1989. 36 í, i SciELO Mem. Inst. Butantan, 52 (supl.l, 1990 LOCAL PATHOLOGICAL EFFECTS INDUCED BY BOTHROPS SNAKEVENOMS José Maria Gutierrez, Instituto Clodomiro Picado, Facultad de Microbiologia, Universidad de Costa Rica, San José, Costa Rica. Envenomations caused by snakes of the genus Bothrops induce conspicuous local tissue damage. This effect, which appears soon after venom injection, is characterized by myonecrosis, hemorrhage and edema. Muscle necrosis is due to (a) the action of myotoxins, some of which have phospholipase A 2 structure. Myotoxins affect the integrity of skeletal muscle plasma membrane by a mecha- nism not directly related to phospholipid hydrolysis. (b) The ischemia that deve- lops in muscle tissue as a consequence of drastic vasculature damage, i.e. hemorrhage and arterial lesions. A portion of necrotic muscle regenerates after venom induced damage. However, in many cases muscle regeneration is poor, mainly due to the alteration of muscle microvasculature. Hemorrhage is caused by the action of metalloproteases which probably de¬ grade collagen and other components of the basal lamina of capillary vessels. As a consequence, capillaries are disrupted and hemorrhage occurs. It is also possible that hemorrhagic toxins affect directly the endothelial cells of capilla¬ ries, although this has not been clearly established. There is a conspicuous im- munologic cross-reactivity of hemorrhagic toxins in Bothrops venoms, and antivenoms usually have high neutralizing ability against these toxins when tes- ted by the traditional preincubation assay. However, when antivenoms are admi- nistered after venom injection, neutralization of hemorrhage and myonecrosis is only partial, probably due to the rapid development of these effects once venom is injected. Local edema is a typical manifestation of envenomations by Bothrops snakes. It is probably caused by a combination of elements such as: direct effect of ve¬ nom on vessels and release of endogenous mediators like histamine, kinins and prostaglandins due to the action of venom components on mast cells, kininogens and phospholipids, respectively. In some cases, edema is responsible for eleva- tion of interstitial hydrostatic pressure in muscle compartments, which might re- 37 cm 2 3 z 5 6 11 12 13 14 15 16 Mem. Inst. Butantan, 52 (supl.), 1990 sult in a compartmental syndrome. Antivenoms are usually of low efficacy in neu- tralizing edema. Thus, new pharmacological approaches must be introduced to deal with this relevant effect. Besides their action on muscle cells and microvasculature, Bothrops venoms also affect arteries, inducing thrombosis and damage to the arterial walls. Arte¬ rial damage, in turn, causes ischemia and further muscle necrosis. In addition, these venoms affect intramuscular nerves. This effect may be relevant for mus¬ cle regeneration, since intact innervation is a basic requirement for skeletal mus¬ cle regeneration. Acknowledgements:Pans of this research have been supported by University of Costa Rica, International Foundation for Science and the Third World Academy of Sciences. 38 cm SciELO 10 11 12 13 14 15 16 Mem. Inst. Butantan, 52 (supl.), 1990 HEMATOLOGICAL DISTURBANCES INDUCED BY BOTHROPS VENOM Ida S. Sano-Martins, Seção de Hematologia, Instituto Butan¬ tan. São Paulo. Local and systemic disturbances are usually observed in patients bitten by Bothrops snakes. The most common systemic disturbance is blood incoagulability 9 occuring more frequently in patients who were bitten by young snakes. 6 This incoagulability is attributed mainly to fibrinogen consumption in- duced by thrombin-like fractions. 8 In addition to these fractions some Bothrops venoms may activate factor X and II 3 causing formation of intravascular throm- bin. 1 This is confirmed by the presence of cross-linked fibrin fragment D in blood from patients. 2 Besides, the intravascular coagulation causes an activation of the fibrinolytic system. Moreover, a mild thrombocytopenia is observed which may be induced either by the activated coagulation system or by components from Bothrops venom acting on platelets such as botrocetin, which has agglutinant activity 5 and thrombocytin which has aggregating activity 4 . The role of all these fractions in envenomations is still uncertain, although the in vitro activities are well studied. In addition to the action on hemostasis most of these venoms possess an in vitro hemolytic activity 7 . However, there are no descriptions of serious intravas¬ cular hemolysis in patients. The action of these venoms on other blood cells seems to be unspecific. In conclusion, the most specific and apparent hematological disturbance in Bothrops envenomations is the blood incoagulability and throm¬ bocytopenia. REFERENCES 1. KAMIGUTI, A.S.; MATSUNAGA, S.; SPIR, M.; SANO-MARTINS, I.S.; NAHAS, L. Al- terations of the blood coagulation system after accidental human inoculation by Bothrops jararaca ve nom. Braz. J. Med. Biol. Res., 79:119-204, 1986. 2. MARUYAMA, M.; KAMIGUTI, A.S.; CARDOSO J.L.C.; SANO-MARTINS, I.S.; CHUD- ZINSKI, A.M.; SANTORO, M.L.; MORENA, P.; TOMY, S.C.; ANTONIO, L.C.; Ml- HARA, H.; KELEN, E.M.A. Studies on blood coagulation and fibrinolysis in patients bitten by Bothrops jararaca (jararaca). Thrombos. Haemost., 63: 449-453, 1990. 3. NAHAS, L.; KAMIGUTI, A.S.; BARROS, M.A.R. Thrombin-like and factor X-activator components of Bothrops snake venoms. Thrombos. Haemost., 41: 314-328, 1979. 4. NIEWIAROWSKI, S.; KIRBY, E.P.; STOCKER, K. Thrombocytin: a novel platelet activating 39 cm 2 3 z 5 6 11 12 13 14 15 16 Mem. Inst. Butantan, 52 (supl.), 1990 enzyme from Bothrops atrox venom. Thrombos. Res., 70:863-869, 1967. 5. READ, M.S.; SHERMER, R.W.; BRINKHOUS, K.M. Venom coagglutinin: an activator of platelet aggregation dependent on von Willebrand factor. Proc. Natl. Acad. Sei. USA, 75: 4514-4518, 1978. 6. RIBEIRO, L.A. & JORGE, M.T. Alteração do tempo de coagulação sangüínea em pa¬ cientes picados por serpentes Bothrops jararaca adulto e filhote. Rev. Hosp. Clín. Fac. Med. São Paulo, 44: 143-145, 1989. 7. ROSENFELD, G.; KELEN, E.M.A.; NUDEL, F. Flemolytic activity of animal venoms. I. Classification in different types and activities. Mem. Inst. Butantan, 30: 117-132, 1960/62. 8. ROSENFELD, G.; NAFIAS, L.; KELEN, E.M.A. Coagulant, proteolytic and hemolytic properties of some snake venoms. In: BüCHERL, W.; BUCKLEY, E.; DEULOFEU, V. ed. Venomous animais and their venoms. New York, Academic Press, 1967, p. 229-273. 9. ROSENFELD, G. Symptomatology, pathology and treatment of snake bites in South America. In: BüCFIERL, W.; BUCKLEY, E.; DEULOFEU, V. ed. Venomous animais and their venoms. New York, Academic Press, 1971. p. 345-384. 1, | SciELO Mem. Inst. Butantan, 52 (supl.), 1990 SYSTEMIC ACTIVITIES OF BOTHROPIC VENOMS Júlia Prado Franceschi, Department of Pharmacology, Faculty of Medicai Sciences, State University of Campinas, São Paulo. Bothropic venoms can affect animais both indirectly, through the liberation of pharmacologically active substances and directly through their action on cellular membranes. 11 Among the many enzymatic activities presented by bothropic venoms only a few are important in the genesis of pharmacological effects namely proteolytic, coagulant, phospholipasic and hyaluronidasic activities. The majority of bothropic venoms exhibit in vitro coagulant activities 1 - 2 - 8 - 4 ; in vivo they can produce hypercoagulability followed by incoagulability through fibrigonen consumption. We investigated 5 - 6 the effects of B. jararaca and. B. erythromelas venoms on arterial blood pressure of anaesthetized dogs following intravenous injection. The doses used were 100 nglkg for B. jararaca and 25 /xg/kg for B. erythromelas. With this dose B. erythromelas venom caused death in 60% of the injected animais, while no deaths were observed with B. jararaca venom. A fali of mean arterial pressure to shock leveis (30 — 40 mm Hg) was observed in all survivors. Besides this effect we also observed thrombocytopenia and slight hemoconcen- tration. In the case of B. erythromelas venom (but not of B. jararaca venom) a brief tachypnea was followed by bradypnea and in some animais, by an apnea which was not counteracted by section of both vagi. Artificial respiration failed to maintain blood pressure. Pretreatment with Metronidazole (125 mg/kg) was able to prevent the onset of shock and the thrombocytopenia induced by i.v. injection of B. jararaca venom 5 . This latter effect could be attributed to its anti-aggregating properties 6 . Heparin (200 IU/kg) and cellulose sulfate (40 mg/kg) protected the dogs against the lethal effects of B. erythromelas venom, but did not affect significantly either the fali of arterial blood pressure or thrombocytopenia. Pretreatment with mepyra- mine also failed to prevent the onset of shock suggesting that histamine release is not a major factor. Bradykinin formation 7 may be involved as disappearance of circulating kinino- gen has been reported following the injection of B. jararaca venom in rats 9 . The liberation of EDRF is supported by the finding that pretreatment with an EDRF antagonist, nitro methylate arginine, 10 reduced the fali of blood pressure and pro- 41 cm 2 3 z 5 6 11 12 13 14 15 16 Mem. Inst. Butantan, 52 (supl.), 1990 moted recovery following i.v. injection of B. erythrometas venom. However, the coagulant activity remained unchanged. Multiple hemorrhagic foci were present in all animais studied. Hemorrhagins have been detected in most bothropic venoms and could worsen the shock, not only by their direct action on collagen IV 3 but also by inhibiting the platelet aggre- gation induced by ADP. The inhibition of thrombin-induced platelet aggregation was one more detectable effect of B. erythromelas venom. Many factors contribute to the genesis of shock following bothropic enveno- mation. REFERENCES 1. BRAZIL, V. & RANGEL PESTANA, B. Nova contribuição ao estudo do envenenamen¬ to ophidico. VI. Ação coagulante. Rev. med. São Paulo, 72:415-425, 1909. 2. BRAZIL, V. & VELLARD, J. Action coagulante et anticoagulante des venins. Ann. Inst. Pasteur, 42:403-451, 1928. 3. MANDELBAUM, F. R. & ASSAKURA, M. T. Antigenic relationship of hemorrhagic fac¬ tors and proteases isolated from the venoms of three species of Bothrops snakes. Toxicon, 26: 379-385, 1988. 4. NAFIAS, L.; KAMIGUTI, A. S.; BARROS, M. A. R. Thrombin-like and factor X-activator components of Bothrops snake venoms. Thrombos. Haemostas., 4 7:314-328, 1979. 5. PRADO-FRANCESCHI, J. (1988). O metronidazol no acidente botrópico, um estudo experimental. In: SIMPÓSIO ANUAL DA ACIESP SOBRE TOXINAS PROTÉICAS, 12. Anais. Campinas, ACIESP, 1988. p. 24. 6. PRADO-FRANCESCHI, J. & ANTUNES, E. Effect of Metronidazole on platelet aggre¬ gation. Braz. J. Med. Biol. Res., 22:1497-1500, 1989. 7. ROCHA E SILVA, M.; BERALDO, W. T.; ROSENFELD, G. Bradykinin, a hypotensive and smooth muscle stimulating principie released from plasma globulin by snake venoms and trypsin. Amer. J. Physiol., 756:261-273, 1949. 8. ROSENFELD, G.; HAMPE, O. G.; KELEN, E.M.A. Coagulant and fibrinolytic activity of animal venoms; determination of coagulant and fibrinolytic index of different spe¬ cies. Mem. Inst. Butantan, 29:143-163, 1959. 9. ROTHSCHILD, A.M. & ALMEIDA, J.A. Role of bradykinin in the fatal shock induced by B. jararaca venom in the rat. In: VRIES, A. & KOCHVA, E. ed. Toxlns of animal and plant origin. New York-London-Paris, Gordon and Breach, 1972. v.2. 10. SUNG, C.P.; ARLETH, A.J.; SHIKANO, K.; BERKOWITZ, B.A. Characterization and function of bradykinin receptors in vascular endothelial cells. J. Pharmacol. Exper. Ther., 247:8-13, 1988. 11. VITAL BRAZIL, O. Peçonhas. In: CORBETT, C.E. ed. Parmacodinâmica. 6. ed. Rio de Janeiro, Guanabara Koogan, 1982. p. 1044. 42 cm SciELO 10 11 12 13 14 15 16 Mem. Inst. Butantan, 52 (supl.), 1990 BOTHROPIC ACCIDENTS João Luiz Costa Cardoso, Hospital Vital Brazil, Instituto Butan¬ tan, São Paulo. Bothropic accidents represent about 90% of snakebite envenomation in South America 7 . In 1989, 20,748 snakebite accidents were notified to the Brazilian Ministry of Health. The majority, 67,3% of the cases were attributed to the genus Bothrops. Of the 20 recognized species 4 are most commonly responsible for human accidents: 6. jararaca, in the South-Southeast; B. moojeni, in the Midwest; B. erythromelas, in the Northeast and B. atrox in the North Brazil. Bothrops atrox is widely distributed in the Amazon region, where over 90% of accidents are at¬ tributed to this species 3 . Rosenfeld 6 introduced simplified diagnostic criteria based on pathophysiolog- ical characteristics of envenomation, which are used up to now. From a medicai point of view, there are three recognized activities responsible for the major characteristics of bothropic envenomation: ''proteolytic'', haemor- rhagic and coagulant. ''Proteolytic” disturbances are the most important clinicai alterations initially characterized by solid oedema and local pain. Blister and necro- sis/ abscess may occur sometimes leading to disabilities. Recent data show that Morganella morgani is identified in over 40% of abscess cultures 2 . Haemorrhagic phenomena such as gengivorhagia and purpurae have been cor- related to the presence of haemorrhagins although their exact implication must be further studied in patients. Coagulation disturbance caused by South American snake venoms have been recently revised. 4 Acute renal failure (ARF) is considered to be the major cause of death in bothrop¬ ic envenomation. In a report of 63 patients with ARF following snakebite, 32 (51 %) were caused by Bothrops while 31 (49%) were caused by Crotalus. Seven pa¬ tients (7/32) bitten by Bothrops in this first group showed renal cortical necrosis 1 . According to the Ministry of Health the lethality rate of snakebite in Brazil is 0,5% 5 . Serumtherapic treatments are still based on empirical criteria. Further studies are needed in order to establish better schedules based on scientific findings. 43 cm 2 3 L 5 6 11 12 13 14 15 16 Mem. Inst. Butantan, 52 (supl.), 1990 REFERENCES 1. AMARAL, C.F.S.; REZENDE, N.A.; SILVA, O.A. da; RIBEIRO, M.M.F.; MAGALHÃES, , R.A.; CARNEIRO, J.G.; CASTRO, J.R.S. Insuficiência renal aguda secundária a acidente botrópico e crotálico. Análise de 63 casos. Rev. Inst. Med. trop. S. Paulo, 28: 220-227, 1986. 2. ANDRADE, J.G.; PINTO, R.N.L.; ANDRADE, A.L.S.; MARTINELLI, C.M.T.; ZICKER, F. Estudo bacteriológico de abscessos causados por picadas de serpentes do gênero Bothrops. Rev. Inst. Med. trop. S. Paulo, 37:363-367, 1989. 3. HAAD, J.J.S. Las serpientes del género Bothrops en la Amazônia Colombiana. Acta med. Colomb., 74:148-165, 1989. 4. KAMIGUTI, A.S. & CARDOSO, J.L.C. Haemostatic changes caused by the venoms of South American snakes. Toxicon, 27:955-963, 1989. 5. MINISTÉRIO DA SAÚDE/SNABS. Acidentes Ofídlcos N° 40, mimeo, 1989. 6. ROSENFELD, G. Symptomatology, pathology and treatment of snake bites in South America. In: BUCHERL, W.; BUCKLEY, E.; DEULOFEU, V. ed. Venomous animais and their venoms. New York, Academic Press, 1971, v. 2. p. 345-384. 7. WORLD HEALTH ORGANIZATION. Progress in the characterization of venoms and stan- dardization of antivenoms. Publication, N° 58. Geneva, 1981. 44 cm SciELO 10 11 12 13 14 15 16 Mem. Inst. Butantan, 52 (supl.). 1990 INTRACRANIAL HAEMORRHAGE AFTER SNAKE BITE João Aris Kouyoumdjian, Faculdade de Medicina, São José do Rio Preto, São Paulo. The occurrence of intracranial haemorrhage after snake bite have been described in a few sporadic cases all over the world. In Brazil one could find six cases of this kind of complication, all as a result of bothropic envenomation. In five cases the bleeding occured into the brain tissue (intracerebral haemorrhage) and in one case was extradural with fatal outcome (table). Pituitary haemorrhage had also been described after Bothrops jararacussu bite followed by acute or chronic pituitary failure. Systemic haemorrhage could occur as a result of defibrinogenation caused by coagulant activity of the venom, but blood incoagulability would not necessarily result in haemorrhage if the endothelial cells of the small blood vessels are preserved. If the venom contained sufficient amounts of the haemorrhagins (which damage the endothelial cells of the small blood vessels) such damage, combined with incoagulable blood would certainly increase the bleeding problem. In four cases reported in Brazil there was blood incoagulability but at least in one case (Kouyoumdjian et a!. 2 ) the clinicál picture from intracranial haemorra- hage started before it, probably caused by haemorrhagins. The diagnosis of intracranial haemorrhage should be suspected in patients that after having been bitten mainly by bothropic snakes become confused with ab- normality at the levei of consciousness and localized neurological signs. The presence of haematoma should be searched for by means of a CT-Scan performed as soon as possible. 45 cm 2 3 L. 5 6 11 12 13 14 15 16 Mem. Inst. Butantan, 52 (supl.), 1990 TEIXEIRA (3) SESSO (4) SESSO (4) BARROS (1) TEKUYA (5) KOUYOUMDJIAN (2) YEAR 1943 1954 1954 1986 1990 1990 SNAKE ACCIDENT bothropic bothropic (B.jararaca) bothropic (B.jararacussu) bothropic bothropic bothropic (B.moojeni) AGE - 21 6 13 SEX - male male male CLINICAL hours 3 days 12 hours 4 days 5 hours PICTURE (time) left hemiplegia left hemiplegia mental confusion unconsciousness decerebratlion frontal headache neck stiffness mental confusion unconsciousness decerebratlion anisocoria coma COAGULATION blood incoagulability blood incoagulability blood incoagulability late blood incoagulability INTRACRANIAL HAEMORRHAGE intracerebral haemorrhage intracerebral haemorrhage right parietal intracerebral haemorrhage right basal nuclei intracerebral haemorrhage left parietal intracerebral haemorrhage right frontal extradural haematoma left frontal OUTCOME death death death right hemiplegia aphasia normal death REFERENCES 1. BARROS, R.S. & JANUARIO, M.C. Hemorragia subaracnoidea após acidente ofídico botrópico. Arq. bras. Neurocirurgia, 5: 253-255, 1986. 2. KOUYOUMDJIAN, J.A.; POLIZELLI, C.; LOBO, S.N.A.; GUIMARÃES, S.M. Fatal ex- tradural haematoma after snake bite (Bothrops moojeni). Trans. roy. Soc. trop. Med. Hyg., (submitted to publication). 3. ROSENFELD, G. Symptomatology, pathology and treatment of snake bites in South America. In: BUCHERL, H.; BUCKLEY, E.; DEULOFEU, V. ed., Venomous animais and their toxins. New York, Academic Press, 1965. p. 345-383. 4. SESSO, J.; WAJCHENBERG, B.L.; CINTRA, A.B.U. Cerebrovascular accidents in Bothrops poisoning. International Conference on Animal Venoms, 1954. 5. TERUYA, K.; MAIA, P.Z.; ZANIN, R.C.S.; SILVA, F.R.S.V. Relato de um caso de hemor¬ ragia intracraniana por acidente botrópico. In: CONGRESSO DA SOCIEDADE BRASILEIRA DE MEDICINA TROPICAL, 26. Acidentes por animais peçonhentos, tema livre, 1990. 6. WOLFF, H. Insuficiência hipofisária anterior por picada de ofídio. Arq. bras. Endocrinot. Metab., 7:25-47, 1958. 46 cm SciELO 10 11 12 13 14 15 16 Mem. Inst. Butantan, 52 (supl.), 1990 SNAKEBITE ACCIDENTS IN GOIÁS Raimundo Nonato Leite Pinto, Hospital de Doenças Tropicais HDT/SUDS-GO, Goiânia, GO. In 1989 the Centro de Informações Tóxico-Farmacológicas of Goiás (CIT/SUDS- GO), reported 1,764 snakebite accidents in the State of Goías. The genus Bothrops was responsible for 57.8% of the accidents, 15.2% by Crotalus, 4.7% by non venomous snakes and, in 22.3% of the cases, the snake was not identified. Most of the accidents where the snake was not identified were treated as caused by a snake of the genus Bothrops, with good clinicai results, suggesting this caus- ing agent. In 10% of the cases the snake was brought with the victim. Accord- ing to the data of geographic distribution of venomous animais by the Núcleo Regional de Ofiologia de Goiânia (NUROG-UCG/MS), the animais most common in the States of Goiás, Tocantins and Distrito Federal are Bothrops moojeni Hoge, Bothrops neuwiedi Wagler, Bothrops alternatus Duméril, Bibron & Duméril, and Crotalus durlssus collilineatus Amaral. The species Bothrops moojeni has the most widespread geographic distribution and the highest frequency. Bothrops neuwiedi shows smaller distribution and lower frequency. Bothrops neuwiedi is represent- ed by the subspecies Bothrops neuwiedi goyazensis, Bothrops neuwiedi mat- togrossensis, and Bothrops neuwiedi pauioensis. Only in a small part of the territory of Goiás, in the south, was the presence of Bothrops alternatus confirmed. The geographic distribution of Crotalus durissus collilineatus is very large and has a high frequency, especially in the middle-north of the State of Goiás, State of Tocan¬ tins and Distrito Federal. In 1,301 cases (77.4%) the accidents involved only male patients. In the age group 13-35 most accidents occurred (49.7%); with 1 7.0% of the accidents occurring between the ages of 0-12 years. In about 16.4% of the cases medicai attention was given within the first hour after the accident, and 62.8% received attention within three hours after the accident. 74.7% of the accidents involved the lower part of the legs (ankles and feet). Among all reported accidents 72.6% occurred between October and April, and the months of June and July (winter) showed the lowest frequency of accidents. The signs/symptoms most frequent in accidents caused by Bothrops were edema (90%), pain (78%), blisters (11%) and local hemorrhage (7.4%). In 85.7% of the cases there was total recovery and in 0.7% the cases resulted in death. In 15.7% of the cases abscesses formed. The aerobic bactéria isolated most frequently 1 , were Morganella morganii (44.4%), Escherichia coli (20.2%), and Providencia sp. (13.2%). In vitro, these bactéria showed high sensitivity to chlo- ranfenicol, which also shows good results in the treatment of anaerobic bactéria isolated from these abscesses. 47 cm 2 3 Z 5 6 11 12 13 14 15 16 Mem. Inst. Butantan, 52 (supl.), 1990 CHEMICAL AND PHARMACOLOGICAL PROPERTIES OF SCORPION AND SPIDER VENOMS Carlos Ribeiro Diniz, Department of Biochemistry, Federal University of Minas Gerais, Belo Horizonte, MG. The aim of the combined Chemical and pharmacological approach in the study of venoms is to identify molecular entities responsible for specific pharmacologi¬ cal effects, to study their mechanism of action and hopefully to contribute for the understanding of the clinicai manifestations and treatment of patients stung by these animais. The description of the symptoms and signs of the envenomation produced in man and experimental animais done by the pioneers of the investigation on scor- pion and spider venoms in South America: Vital Brazil , 7 H.R. Maurano 6 , J. Vel- lard , 12 B. Houssay, M. Physalix , 8 O.Magalhães, 5 , O.M. Campos, 7 , P. Carvalho, A. Barrio \ and others indicated that hardly the effects induced by these ven¬ oms could be explained by presence in the venom of only one active Chemical entity. In the scorpion the targets of the venom action are the excitable tissues of the body and more precisely the ion channels. The protein and peptide fractio- nation methods introduced in the study of venoms by Karl Slotta 11 , and J. Moura Gonçalves 3 allowed us to separate at least ten neuroactive substances in the Tityus serrulatus venom . 4 These neurotoxins are comparable to the toxins iso- lated from the venom of the African scorpion Androctonus australis and to the toxins of North American (México) scorpion Centruroides s. sufusus in its lethali- ty to fly larva, and mice either by s.c. or i.c.v. injection. The results are shown in Table I. The toxicity of Tityus serrulatus venom probably is higher for children . In the venom of the armed spider Phoneutria nigriventer, using the observa- tion of appearance of the different signs and symptoms induced in mice by i.p. injection of P. nigriventer, as classically described by Schenberg and Pereira Lima, 10 , was possible to isolate four highly purified fractions of this venom. One of these fractions is a sodium channel ligand 9 . The fractionation of the venom of Lycosa and Loxosceles has not advanced, but proteases and neurotoxic sub¬ stances were detected in the Lycosa venom. 49 ff 'SciELO 0 11 12 13 14 15 16 I Mem. Inst. Butantan, 52 (supl.), 1990 TABLEI Comparison between the activities of different scorpion venoms and toxins on fly larva and mouse Sample Fly larva: CPU 3 p g/100mg Mouse: LD 50 (s.c.) p g/20g (i.c.v.) ng/20g AaH venom 0.300 (b) 8.40 (b) n.d. TS venom 0.260 17.50 n.d. AaH IT 0.001 (b,c) >1000.00 >50 x 103 1 n.t. (b,c) 0.34 (b) 10.0 II n.t. (b,c) 0.18 (b) 0.5 III n.t. (b,c) 0.45 (b) 7.0 Css II >2.000 0.5 5 Css VI >1.000 0.05 1.7 Ts 1 >1.780 3.50 24.0 II >1.780 3.70 6.0 III >2.800 2.70 80.0 IV >2.800 0.40 24.0 VI 0.091 8.50 2.5 VII 0.046 4.70 0.6 VIII 0.051 7.20 11.0 AaH: Androctonus australis Hector; Css: Centruroides suffusus suffusus; Ts: Tityus serrulatus; n.d. not determined, nt: non-toxic; s.c.: subcutaneous; i.c.v intracerebroventricular; a CPU: contraction paralysis unit, (4); (b,c) according to Zlotkin et ai REFERENCES 1. BARRIO, A. Spastic action of the venom of the spider Phoneutria fera. Acta lat.-amer. Phys., 5: 132-143, 1955. 2. BRAZIL, V. Contribuição ao estudo do envenenamento pela picada de escorpião e seu tratamento. Colet. Trab. Inst. Butantan, S. Paulo, 7:69-81, 1901-1907. 3. GONÇALVES, J.M. & POLSON, A. The electrophoretic analysis of snake venoms. Arch. Biochem., 73:253-262, 1947. 4. LIMA, M.E.; MARTIN, M.F.; DINIZ, C.R.; ROCHAT, H. Biochem. Biophys. Res. Com- mun., 737:296-302, 1986. 5. MAGALHÃES, O. Contribuição para o conhecimento da intoxicação pelo veneno de escorpiões. Mem. Inst. Òswaldo Cruz, 27(11:5-139, 1928. 6. MAURANO, U.R. Do escorpionismo. Rio de Janeiro, Faculdade de Medicina do Rio de Janeiro, 1915. Tese. Tip. do Jornal do Comércio, RJ. 7. MELLO CAMPOS, 0. Os escorpiões brasileiros. Mem. Inst. Oswaldo Cruz, 7 7:67-89, 1924. 8. PHYSALIX, M. Animaux venimeux et venins. Paris, Masson et Cie., 1922. 9. REZENDE JR., L; CORDEIRO, M.N.; DINIZ, C.R. Separation of neurotoxin in the armed spider venom, 1990. (submitted). 10. SCHENBERG, S. Venoms of Ctenidae. In: Bettini, S., ed. Arthropod venoms. New York, Springer, 1978, p.217-245. 11. SLOTTA, K.H. & FRAENKEL-CONRAT, H. Two active proteins from rattlesnake ve¬ nom. Nature, 742:213-214, 1938. 12. VELLARD, J. Le venin des araignés. Paris, Masson et Cie., 1936. 50 cm SciELO 10 11 12 13 14 15 16 Mem. Inst. Butantan, 52 (supl.), 1990 PHARMACOLOGY OF TITYUS SERRULATUS SCORPION VENOM Lineu Freire-Maia, Departamento de Farmacologia. ICB-UFMG. Belo Florizonte, MG. ABSTRACT: The data presented in this lecture seem to indicate that the effects evoked by scorpion toxins, obtained from Tityus serrulatus scorpion venom are due to actions on specific sites of sodium channels, with a subsequent depolari- zation of the membranes. The respiratory arrhythmias are due, at least in part, to depolarizations of visceral afferent fibers, induced by the toxin, whereas the other effects are mainly due to release from nerve endings of Chemical media- tors, such as acetylcholine and catecholamines. KEYWORDS: Scorpion venom; pharmacology. INTRODUCTION Scientists all over the world have been interested in scorpion venoms because of the high incidence of fatal cases of scorpion poisoning. 21 I will describe in this lecture the pharmacology of the venom obtained from the South American scor¬ pion Tityus serrulatus Lutz & Mello 1922. Cardiovascular effects. Intravenous injection of crude venom or purified toxins 29 obtained from T. serrulatus scorpion venom in rats or dogs induced an acute arterial hypertension, due to the release of catecholamines from adrenal glands and postganglionic nerve endings; the catecholamines acting on alpha adrenergic receptors would increase the peripheral resistence; at least, the catecholamines could act also on beta adrenergic receptors, either increasing the cardiac contractility or releasing renin from the kidneys 1 ' 10 ' 35 ' 45 . Scorpion toxin also induces complex cardiac arrhythmias in the rat. Bilateral cervical vagotomy does not prevent the production of sinus bradycardia, SA block or AV block, after toxin injection; in vagotomized rats, physostigmine enhances, 51 cm 2 3 L 5 6 11 12 13 14 15 16 Mem. Inst. Butantan, 52 (supl.), 1990 hexamethonium decreases and atropine abolishes the bradycardiac effects. Moreover, propranolol prevenis or abolishes the sinus tachycardia and changes the ventricular into a sinus rhythm. Based on the data we have concluded that the sinus bradycardia, SA and AV block are due to release of acetylcholine by actions of the toxin on vagai ganglia and postganglionic nerve endings in the heart whereas the sinus tachycardia, the ventricular ectopic beats and the idioventric- ular rhythm are caused by activation of beta adrenergic receptors in the heart by catecholamines released by the toxin. 19 ' 23 The crude venom of the scorpion Tityus serrulatus evokes, in isolated guinea pig heart, a short-lasting bradycardia followed by a conspicuous increase in the force and the rate of cardiac contractions, due to a local release of acetylcholine and noradrenaline 10 . A direct demonstration of the release of noradrenaline in isolated guinea pig atria by tityustoxin, extracted from the venom of Tityus serrulatus 29 was also reported 36 . Experiments performed by our group in isolated guinea pig hearts have also shown that after the initial events elicited by scorpion toxin, oscillations in heart rate, contractile force and coronary flow are observed during a period of 5-15 min. Recording of the electrical activity of the heart shows that the oscillations of rhythm are due to wandering pacemakers; as atropine prevents the appear- ance of such pacemakers it seems likely that they are related to release of acetyl¬ choline by toxin 1 . Our group has also performed experiments with toxin, the most important toxin extracted from T. serrulatus scorpion venom 41 - 43 ' 48 . Injection of toxin in isola¬ ted guinea pig heart evokes complex effects, which could be divided into 3 phases: an initial phase (tachycardia or bradycardia associated with an increase in con¬ tractile force), an intermediate phase (oscillations of the cardiac rhythm) and a third phase (sinus tachycardia). The bradycardia and oscillations of rhythm are cholinergic in nature, whereas the tachycardia and the increase in contractile force are adrenergic in nature 45 ' 40 . According to some authors 32 - 33 there is no value of antivenom in the treat- ment or prevention of the cardiovascular manifestations of scorpion poisoning. Our group does not agree with this statement, based on laboratory and clinicai evidences 6 ' 7 ' 20 ' 21 ' 24 . Recently, we have shown that perfusion of the isolated guinea pig heart with Locke solution containing 2% of Butantan antivenom prevents almost totally the cardiovascular effects induced by crude Tityus serrulatus scorpion venom 45 ' 46 . The treatment of severe cases of scorpion poisoning should be performed in an Intensive Care Unit, consisting of symptomatic measures, support of vital func- tions and neutralization of circulating venom 6 ' 7 ' 21 . Putmonary edema. Many patients stung by scorpions of several species die with pulmonary edema 21 . It seems that the lung edema would be due, at least in part, to a heart failure, caused by an increase in afterload, 2 - 27 a decrease of left ventricular compliance, 34 the presence of a severe sinus tachycardia 10 ' 21 a myocardial damage 40 and an increase of venous return (preload), caused by dis- charge of catecholamines 38 ' 44 . Another mechanism contributing to the genesis of pulmonary edema, following the toxin injection, would be the release of sub- stances which increase the vascular permeability, such as kinins, histamine and prostaglandins 5 ' 27 ' 42 . Respiratory effects. Injections of venoms from several scorpion species producè complex respiratory arrhythmias, such as tachypnea, hyperpnea, periodic respi- ration and apnea 21 Experiments performed by our group have shown that bilater¬ al cervical vagotomy and denervation of carotid bodies prevent the gasping, ataxic 52 cm SciELO 10 11 12 13 14 15 16 Mem. Inst. Butantan, 52 (supl.), 1990 and periodic respiration induced by the toxin 22 . Moreover, local anesthesia of the cervical vagus nerves with lidocaine abolishes the apnea induced by the toxin 24 . These experiments seem to indicate that the respiratory arrhythmias evoked by scorpion toxin are reflex in nature. To test such a hypothesis our group recorded the action potentials of the vagus and phrenic nerves, simultaneously. The ap¬ nea evoked by scorpion toxin was accompanied by suppression of phrenic nerve activity, while the action potentials in the vagus nerves were increased in ampli¬ tude and/or frequency. Anesthesia of the vagus nerves with lidocaine or bilateral vagotomy suppressed the vagus nerve potentials which were present during the apnea and reestablished the phrenic nerve activity. Based on these data, we have concluded that the apnea elicited by scorpion toxin is due to a reflex mechanism, evoked by stimulation of vagai afferent fibers 21 . Effects on the neuromuscular junction. Injection of Tityus serrulatus scorpion venom induces the release of an acetylcholine-like substance from the inervated but not from the denervated rat diaphragm 49 . The release of this substance could account for the twitches observed and also for the decurarizing activity of scorpi¬ on venom 14 . On the other hand, electrophysiological studies on the action of tityustoxin, purified from the venom of Tityus serrulatus, on rat phrenic nerve- diaphragm muscle preparation, have shown that the toxin has a presynaptic and postsynaptic action at the neuromuscular junction 50 . Effects on the gastrointestinal System. An intense sialagogue effect, induced by T. serrulatus scorpion venom in mice, is abolished by atropine, being, there- fore, cholinergic in nature 15 . Our group has studied the effect induced by a pu¬ rified scorpion toxin in rats. We have shown that the toxin increases the flow, the kallikrein and amylase secretions. Pharmacological tests have shown that the increased salivary secretion induced by scorpion toxin in rats is due to choliner¬ gic and adrenergic mechanisms 8 . Our group has shown that an intravenous injection of a toxin obtained from T. serrulatus scorpion venom, in anesthetized rats, evokes a dramatic increase in volume, acid and pepsin output of gastric juice and a significant decrease in its pH. Acute bilateral cervical or abdominal vagotomy does not prevent the gas¬ tric secretion elicited by toxin, whereas atropine or cimetidine abolish partially or totally the toxin effects 12 ' 31 . Recently, we have shown that scorpion toxin in¬ duces the release of acetycholine and histamine from rat stomach 13 . Based on these results, we think that scorpion toxin exerts its action upon the rat stomach by releasing acetylcholine directly from its nerve endings Stores and by releasing histamine directly or indirectly from histaminocytes. Release of gastrin from phylor- ic antrum could also contribute to the overall response in vivo (Cunha-Meio, per- sonal communication). Injection of Tityus trinitatis scorpion venom induces an increase in both volume and amylase secretion from the pancreas of anesthetized dogs 3 . Our group has studied the effects of a toxin obtained from Tityus serrulatus on pancreatic secre¬ tion, in anesthetized rats. The intravenous injection of toxin causes a striking in¬ crease in flow rate, protein content, kallikrein and amylase activities of the pancreatic juice. Sub-diaphragmatic bilateral vagotomy does not prevent the pan¬ creatic secretion induced by toxin, but pre-treatment of the rats with atropine blocks the secretion evoked by toxin. We have presented the hypothesis that the pancreatic secretion induced by scorpion toxin is due to actions of acetylcholine, released from postganglionic nerve fibers, on muscarinic receptors 39 . This hypothesis is supported by in vitro experiments 28 . Venoms from scorpions of the genus Tityus induce acute pancreatitis in laboratory animais 4 ' 37 . 53 cm 2 3 L 5 6 11 12 13 14 15 16 Mem. Inst. Butantan, 52 (supl.), 1990 Incubation of segments of guinea pig ileum with Tityus serrulatus scorpion ven- om releases an acetylcholine-like substance which would be responsible by the contraction of the ileum evoked by the venom 16 . However, our group has shown that atropine does not prevent totally the contraction evoked by the toxin either in rat or guinea pig ileum. 11 ' 26 But we have also shown that the toxin releases acetylcholine from rat and guinea pig ileum, through stimulation of sodium channels 18 ' 26 . We have postulated, then, that the contraction of intestinal smooth muscle elicited by scorpion toxin is due to release of acetylcholine and another Chemical mediator. Based on indirect evidences we presented the hypothesis that this second mediator could be substance P 11 . Release of noradrenallne and acetylcholine by scorpion toxins. Many investi- gators have shown that Tityus serrulatus scorpion venom or one of its purified toxins (tityustoxin) release noradrenaline and acetylcholine in several preparations, and that blockade of sodium channels with tetrodotoxin prevents their release 17 ' 30 ' 47 . It seems likely that scorpion toxin binds to a site different from the tetrodotoxin-binding site, but its physiological role (e.g. release of acetylcho¬ line) depends on an action on sodium-conducting pore of the channel 9 ' 18 ' 26 RESUMO: Os resultados apresentados nesta conferência parecem indicar que os efeitos induzidos pelas toxinas de escorpião, obtidas do veneno de Tityus ser¬ rulatus, são devidos a ações em sítios específicos dos canais de sódio, com despolarização subseqüente das membranas. As arritmias respiratórias são devi¬ das, pelo menos em parte, a despolarizações de fibras aferentes viscerais, induz¬ idas pela toxina, enquanto que os outros efeitos descritos são devidos principalmente a liberação, pelas terminações nervosas, de mediadores quími¬ cos como a acetilcolina e catecolaminas. UNITERMOS: Veneno de escorpião; farmacologia. ACKNOWLEDGEMENTS The author thanks CNPq (Brazil) for a fellowship and Mrs. Maria Celia Silva Costa for typing the manuscript. REFERENCES 1. ALMEIDA, A.P.; ALPOIM, N.C.; FREIRE-MAIA, L. Effects of a purified scorpion toxin (tityustoxin) on the isolated guinea pig heart. Toxicon, 20:855-865, 1982. 2. AZEVEDO, A.D.; SILVA, A.B.; CUNHA-MELO, A.B.; FREIRE-MAIA, L. Cardiovascular and respiratory effects induced by a purified scorpion toxin (tityustoxin) in unanesthe- tized rats. Toxicon, 27.753-759, 1983. 3. BARTHOLOMEW, C.; MURPHY, J.J.; FITZGERALD, O.; McGEENEY, K.F. Action of Tityus trinitatis venom on the canine pancreas. Toxicon, 73:87, 1975. 4. BARTHOLOMEW, C.; MURPHY, J.J.; FITZGERALD, O.; McGEENEY, K.F. Experimen¬ tal studies on the etiology of acute scorpion pancreatitis. Brit. J. Surg., 63:807-810, 1976. 5. BERALDO, W.T. & DIAS DA SILVA, W. Release of histamine by animal venoms and bacterial toxins. In: EICHLER, 0. & FARAH, A. ed. Handbook of Experimental Phar- macology. Berlin, Springer-Verlag, 1966, v. 18. p. 334-366. í, | SciELO 54 Mem. Inst. Butantan, 52 (supl.), 1990 6. CAMPOS, J.A.; SILVA, O.A.; LOPEZ, M.; FREIRE-MAIA, L. Signs, symptoms and treat- ment of severe scorpion sting in children. Toxicon, 77 (suppl.1): 19, 1979. 7. CAMPOS, J.A.; SILVA, O.A.; LOPEZ, M.; FREIRE-MAIA, L. Signs, symptoms and treat- ment of severe scorpion poisoning in children. In: EAKER, D & WADSTROM, T. Natural Toxins. Oxford, 1980. p. 61-68. 8. CATANZARO, O.L.; SANTOS, R.A.S.; PARRA, O.M.; SANTOS, R.M.; FREIRE-MAIA, L.; BERALDO, W.T. Effect of scorpion toxin (tityustoxin, TsTX) on the salivary gland of the rat, in vivo. Agents Actions, 8: 119-124, 1978. 9. CATTERALL, W.A. Cooperative activation of action potential Na + ionophore by neu- rotoxins. Proc. Natl. Acad. Sei. USA, 72: 1782-1786, 1975. 10. CORRADO, A.P.; ANTONIO A.; DINIZ, C.R. Brazilian scorpion venom (Tityus serruia- tus), an unusual sympathetic postganglionic stimulant. J. Pharmac. Exp. Ther., 764:253-258, 1968. 11. CUNHA-MELO, J.R.; FREIRE-MAIA, L.; TAFURI, W.L.; MARIA, T.A. Mechanism of actions of purified scorpion toxin on the isolated rat intestine. Toxicon, 7 7:81-84, 1973. 12. CUNHA-MELO, J.R.; GONZAGA, H.M.S.; ALZAMORA, F.; FREIRE-MAIA, L. Effetcs of purified scorpion toxin (tityustoxin) on gastric secretion in the rat. Toxicon, 2 7:843-838, 1983. 13. CUNHA-MELO, J.R.; ALMEIDA, A.P.; GONZAGA, H.M.S.; GOMEZ, M.V.; FREIRE- MAIA, L. Effect of scorpion toxin on gastric histamine and acetylcholine content in the rat. Braz. J. Med. Biol. Res., 20:393-401, 1987. 14. DEL POZO, E.C. & ANGUIANO, L.G. Acciones dei veneno de alacran sobre la actividad motora dei músculo estriado. Rev. Inst. Saiub. Enferm. trop., 8: 231-263, 1947. 15. DINIZ, C.R. & VALERI, V. Effects of a toxin present in a purified extract of telson from the scorpion Tityus serruiatus on smooth muscle preparations and in mice. Archs. Int. Pharmacodyn., 77:1-12, 1959. 16. DINIZ, C.R. & TORRES, J.M. Release of an acetylcholine-like substance from guinea- pig ileum by scorpion venom. Toxicon, 5: 227-236, 1968. 17. DINIZ, C.R.; PIMENTA, A.F.; COUTINHO-NETTO, J.; POMPOLO, S.; GOMEZ, M.V.; BOHM, G.M. Effect of scorpion venom from Tityus serruiatus (Tityustoxin) on the acetylcholine release and fine strueture of the nerve terminais. Experientia (Basel), 30: 1304-1305, 1974. 18. FREIRE-MAIA, L. Neurotoxins as tools for physiological investigation. Physiologist., 26: 381-390, 1983. 19. FREIRE-MAIA, L. & DINIZ, C.R. Pharmacological action of a purified scorpion toxin in the rat. Toxicon, 8: 132, 1970. 20. FREIRE-MAIA, L. & CAMPOS, J.A. Response to the Letter to the Editor by Gueron and Ovsyshcher — On the treatment of the cardiovascular manifestations of scor¬ pion envenomation. Toxicon, 25: 125-130, 1987. 21. FREIRE-MAIA, L. & CAMPOS, J.A. Pathophysiology and treatment of scorpion poison¬ ing. In: OWNBY, C.L. & ODELL, G.V. ed. Natural Toxins; characterization, phar- macology and therapeuties. Oxford, Pergamon Press, 1989, p. 139-159. 22. FREIRE-MAIA, L.; AZEVEDO, A.D.; COSTA VAL, V.P. Respiratory arrhytmias produced by purified scorpion toxin. Toxicon, 7 7:255-257, 1973. 23. FREIRE-MAIA, L.; PINTO, G.I.; FRANCO, I. Mechanism of the cardiovascular effects produced by purified scorpion toxin in the rat. J. Pharmacol. exp. Ther., 785:207-213, 1974. 24. FREIRE-MAIA, L.; AZEVEDO, A.D.; LIMA, E.G. Pharmacological blockade of the cardi¬ ovascular and respiratory effects produced by tityustoxin in the rat. In: OHSAKA, A.; HAYASHI, K.; SAWAI, Y. ed. Animal, plant, and microbiai toxins. New York, Plenum, 1976 a. V.2. p. 287-298. 25. FREIRE-MAIA, L; CUNHA-MELO, J.R.; FUTURO-NETO, H.A.; AZEVEDO, A.D.; WEIN- BERG, J. Cholinergic and adrenergic effects of tityustoxin. Gen. Pharmac., 7:115-121, 1976b. 55 cm 2 3 z 5 6 11 12 13 14 15 16 Mem. Inst. Butantan, 52 (supl.), 1990 26. FREIRE-MAIA, L; CUNHA-MELO, J.R.; GOMEZ, M.V.; TAFURI, W.L.; MARIA, T.A.; CALIXTO, S.L.; FUTURO-NETO, FI.A. Studies on the mechanism of action of tityustoxin. In: OFISAKA, A.; FIAYASFII, K.; SAWAI, Y. ed. Animal, plant, and microbial toxins. New York, Plenum, 1976c. v.2. p. 273-285. 27. FREIRE-MAIA, L; ALMEIDA, H.O.; CUNHA-MELO, J.R.; AZEVEDO, A.D.; BARRO¬ SO, J. Mechanism of the pulmonary edema Induced by intravenous injection of scor- pion toxin in the rat. Agents Actions, 8:113-118, 1978. 28. GALLAGHER, S.; SANKARAN, H.; WILLIAMS, J.A. Mechanism of scorpion toxin- induced enzyme secretion in rat pancreas. Gastroenterology, 80: 970, 1981. 29. GOMEZ, M.V. & DlNIZ, C.R. Separation of toxin components from the Brazilian scor¬ pion - Tityus serrulatus - venom. Mem. Inst. Butantan, 33:899-902, 1966. 30. GOMEZ, M.V.; DAI, M.E.M.; DlNIZ, C.R. Effect of scorpion venom, tityustoxin, on the release of acetylcholine from incubated slices of rat brain. J. Neurochem. 20:1051-1061, 1973. 31. GONZAGA, H.M.S.; ALZAMORA, F.; CUNHA-MELO, J.R.; FREIRE-MAIA, L. Gastric secretion induced by scorpion toxin. Toxicon, 7 7:316-318, 1979. 32. GUERON, M. & OVSYSHCHER, I. Cardiovascular effects of scorpion venoms. In: Tu, A.T. ed. Handbook of Natural Toxins. New York, Marcei Dekker, 1984. V. 2. p. 639-657. 33. GUERON, M. & OVSYSCHER, I. Letter to the Editor - What is the treatment for the cardiovascular manifestations of scorpion envenomation? Toxicon, 25: 121-124, 1987. 34. GUERON, M., ADOLPH, R.J.; GRUPP, I.L., GABEL, M.; GRUPP, G.; FOWLER, N.O. Hemodynamic and myocardial consequences of scorpion venom. Amer. J. Cardi- ol., 45: 979-986, 1980. 35. LA GRANGE, R.G. Elevation of blood pressure and plasma renin leveis by venom from scorpions, Centruroides sculpturatus and Leiurus quinquestriatus. Toxicon, 75:429-433, 1977. 36. LANGER, S.Z.; ADLER-GRASCHINSKY, E.; ALMEIDA, A.P.; DINIZ, C.R. Prejunction- al effect of a purified toxin from the scorpion Tityus serrulatus. Naunyn- Schmiedeberg's Arch. Pharmac., 287: 243-259, 1975. 37. MACHADO, J.C. & SILVEIRA FILHO, J.F. Indução de pancreatite hemorrágica aguda no cão por veneno escorpiônico de T. serrulatus. Mem. Inst. Butantan, 40/4 7:1-9, 1976/77. 38. MOSS, J.; KAZIC, T.; HENRY, D.P.; KOPIN, I.J. Scorpion venom-induced discharge of catecholamines accompanied by hypertension. Brain Research, 54: 381-385, 1973. 39. NOVAES, G.; CATANZARO, O.L.; BERALDO, W.T.; FREIRE-MAIA, L. Effect of puri¬ fied scorpion toxin (tityustoxin) on the pancreatic secretion of the rat. Toxicon, 20:847-853, 1982. 40. POON-KING, T. Myocarditis from scorpion stings. Brit. med. J., 1:374-371, 1963. 41. POSSANI, L.D.; ALAGON, A.C.; FLETCHER JR., P.L.; ERICKSON, B. Purification and properties of mammalian toxins from the venom of the Brazilian scorpion Tityus serrulatus Lutz and Mello. Archs. Biochem. Biophys., 780:394-403, 1977. 42. ROTHSCHILD, A.M. & CASTANIA, A. Contribution of vasopressor and plasma kinino- gen changes towards acute adrenaline pulmonary edema in the rat. Naunyn- Schmiedeberg’s Arch. Pharmac., 295:177-181, 1976. 43. SAMPAIO, S.V.; LAURE, C.J.; GIGLIO, J.R. Isolation and characterization of toxic proteins from the venom of the Brazilian scorpion Tityus serrulatus. Toxicon, 27:265-277, 1983. 44. SAVINO, E.D. & CATANZARO, O.L. The effects of tityus toxin on the isolated rat por¬ tal vein. IRCS. med. Sei., 7: 317, 1979. 45. SILVEIRA, N.P.; BORBA, V.C.; MORAES E SANTOS, T.; FREIRE-MAIA, L. Estudo com¬ parativo dos efeitos induzidos pelo veneno do escorpião Tityus serrulatus e de duas de suas toxinas purificadas sobre o coração isolado de cobaia. In: REUNIÃO ANU¬ AL DA FESBE, 4. Resumos. Caxambu, 1989. P. 114. 56 cm SciELO 10 11 12 13 14 15 16 Mem. Inst. Butantan, 52 (supl.), 1990 46. SILVEIRA, N.P.; MORAES-SANTOS, T.; AZEVEDO, A.D.; FREIRE-MAIA, L. Effects of Tityus serrulatus scorpion venom and one of its purified toxins (toxin) on the iso- lated guinea pig heart. Comp. Biochem. Physiol. - in press (1990). 47. TAZIEFF-DEPIERRE, F. & ANDRILLON, P. Secretion d'acetylcholine provoquee par le venin de scorpion dan 1'ileon de cobaye et sa suppresion par la tetrodotoxin. C.R. Acad. Sc. (Paris), 276:1631-1633, 1973. 48. TOLEDO, D. & NEVES, G.A. Purification and partial characterization of a second toxin from the scorpion Tityus serrulatus. Comp. Biochem. Physiol., 55B: 249-253, 1976. 49. VITAL BRAZIL, O.; NEDER, A.C.; CORRADO, A.P. Effects and mechanism of action of Tityus serrulatus venom on skeletal muscle. Pharmacol. Res. Comm., 5: 137-150 1973. 50. WARNICK, J.E.; ALBUQUERQUE, E.X.; DINIZ, C.R. Electrophysiological observations on the action of purified scorpion venom, tityustoxin, on nerve and skeletal muscle of the rat. J. Pharmac. Exp. Ther., 796:155-167, 1976. 57 cm JSciELO 0 11 12 13 14 15 16 Mem. Inst. Butantan, 52 (supl.), 1990 PHARMACOLOGY OF PHONEUTRIA VENOM Marcos Dias Fontana, Department of Pharmacology, Faculty of Medicai Sciences, State University of Campinas, São Paulo. Phoneutria nigriventer and P. keyserling (Ctenidae, Labidognata) are very agres- sive wandering solitary spiders. They are responsible for most human accidents of araneism in Center East and South of Brazil. Their venoms are neurotoxic and very potent. 1 ' 3 - 5 The signs and symptoms evoked by them in animais or observed in human accidents are excruciating pain irradiating from the site of introduction, painful cramps, tremors, tonic convulsions, spastic paralysis, sialorrhea, sudore- sis, priapism, tachycardia, arrhytmias and visual disturbances. 1 ' 2 ' 4 The venoms do not produce local edema or necrosis nor induce blood coagulation or hemoly- sis. 1 The mechanism of action of P. nigriventer venom was investigated in our laboratory 5 ' 7 at the isolated rat diaphragm and the isolated guinea pig auricles. In both preparations it was found that the venom produces effects by activating the sodium channels. In the rat diaphragm the venom induced depolarization of the muscle fibre membrane. This effect was abolished by tetrodotoxin (TTX) or by reduction of Na + concentration in the bath fluid. The venom induced increase of the frequency of the miniature end plate potentials (m.e.p.p.) was also abolished by TTX. This last result shows that the sodium channels of the motor nerve ter¬ minal membrane are also activated by the venom. In the isolated guinea-pig auricles, TTX in concentration that reduces only slight- ly (23.6 ± 6.2%) the tension of the muscle fibres abolished the remarkable ef¬ fects of the venom on their chronotropism and inotropism. This shows that the venom acts by activating the sodium channels of the autonomic nerve endings in the auricles, thereby releasing acetylcholine and norepinephrine. Phtx 2 , a toxin of 5,000 daltons isolated from P. nigriventer venom by Diniz is about three times as lethal for mice as the venom (venom LD50 i.v. 377 /xg/kg; PhTx 2 LD50 i.v. 130 /ig/kg). It activates also the sodium channel being about 18 and 2 times as active as the venom in the rat diaphragm and in the isolated guinea pig auricles respectively. 6 REFERENCES 1. BRAZIL, V. & VELLARD, J. Contribuição ao estudo do veneno das aranhas. Mem. Inst. Butantan, 2:5-77, 1925. 2. BRAZIL, V. & VELLARD, J. Contribuição ao estudo do veneno das aranhas. Mern. Inst. Butantan, 3: 243-299,1926. 59 cm 2 3 L 5 6 11 12 13 14 15 16 Mem. Inst. Butantan, 52 (supl.), 1990 3. BUCHERL, W. Novo processo de obtenção de veneno seco, puro, de Phoneutria nigriventer (Keyserling 1881) e titulação da DL50 em camundongos. Mem. Inst. Butantan, 25:153-174, 1953. 4. SCHENBERG, S. & PEREIRA LIMA, F.A. Pharmacology of the polypeptides from the venom of the spider Phoneutria fera. Mem. Inst. Butantan, 33: 627-638, 1966. 5. FONTANA, M.D. & VITAL BRAZIL, O. Mode of action of Phoneutria nigriventer spider venom atthe isolated phrenic nerve-diaphragm of the rat. Braz. J. Med. Biol. Res., 78:557-565, 1985. 6. FONTANA,M.D.; VITAL BRAZIL, O.; DINIZ, C.R. Unpublished results. 7. VITAL BRAZIL,O.; BERNARDO LEITE, G.; FONTANA, M.D. Modo de ação da peçonha da aranha armadeira Phoneutria nigriventer (Keyserling 1891), nas aurículas isola¬ das de cobaia. Ciênc. e Cult., 40:181-185, 1988. 60 cm SciELO 10 11 12 13 14 15 16 Mem. Inst. Butantan, 52 (supl.), 1990 ACCIDENTS WITH SPIDERS OF THE PHONEUTRIA GENUS Fábio Bucaretchi, Centro de Controle de Intoxicações, Facul¬ dade de Ciências Médicas, Universidade Estadual de Campi¬ nas (UNICAMP). Campinas, São Paulo. The spiders of the genus Phoneutria (Perty 1893) are, probably, responsible for most of the spider accidents in human beings in Brazil. 4 In the State of São Paulo, where the accidents are notified with higher frequency, they occur mostly in the beginning of the cold season, in the months of April and May, during the daytime, and inside the residences. 2 ' 5 Experimental studies demonstrated that the venom acts basically on the sodi- um channel, inducing depolarization of muscular fibers and sensitive, motor and autonomic nervous endings. 3 ' 6 Only local signs and symptoms are observed in most of the accidents. Pain is the most frequent symptom and usually develops immediately after the sting, which is variable in intensity, and may radiate to the proximal segment, or may be followed by local sweating, edema, hiperemia, paresthesia and local muscu¬ lar fasciculation. More rarely, especially in children and older patients, systemic manifestations may be found, being reported: generalized sweating, hipothermia, vomiting, sialorrhea, visual acuity disturbances, lacrimation, rinorrhea, tachycardia, cardiac arrythmias, arterial hypotension, priapism, and more rarely, diarrhea, shock, acute pulmonary edema and cardiorespiratory arrest. 1 ' 2 ' 7 The prognosis is good and deaths very rare. There are 8 reports of death in the Brazilian litera- ture since 1 926. REFERENCES 1. ALBUQUERQUE, R.P.; FRANÇA, F.O.S.; ROCHA, A.S.; CARDOSO, J.L.C. Manifesta¬ ções autonômicas de longa duração em paciente picado por Phoneutria sp. In: CON¬ GRESSO DA SOCIEDADE BRASILEIRA DE MEDICINA TROPICAL, 26. Anais. Natal, 1990. Tema Livre 84. 2. BUCARETCHI, F.; VIEIRA, R.J.; ZAMBRONE, F.A.D.; FONSECA, M.R.C.C.; BUSTORFF, J. Avaliação clínico-epidemiológica do foneutrismo na Região de Campinas-SP. In: CONGRESSO BRASILEIRO DE TOXICOLOGIA, 6. Anais. São Paulo, 1989. Tema Livre 5.27. 3. FONTANA, M.D. & VITAL BRAZIL, O. Mode of action of Phoneutria nigriventer spider venom at the isolated phrenic nerve-diaphragm of the rat. Braz J. Med. Biol. Res., 78:557-565, 1985. 61 cm 2 3 L 5 6 11 12 13 14 15 16 Mem. Inst. Butantan, 52 (supl.), 1990 4. MINISTÉRIO DA SAÚDE. Secretaria Nacional de Ações Básicas de Saúde. Acidentes araneídicos: Contribuição ao estudo de morbidade. Boletim n° 22, 1988. 5. RIBEIRO, L.A.; TANUS, M.J.; CARDOSO.J.L.C.; PIESCO, R.V. Acidentes causados por picada de aranha do gênero Phoneutría SP —- 543 casos: aspectos clínicos e epidemi- ológicos. In: CONGRESSO DA SOCIEDADE BRASILEIRA DE MEDICINA TROPICAL, 20. Anais. Salvador, 1984. Tema Livre 304. 6. VITAL BRAZIL, 0.; BERNARDO LEITE, G.; FONTANA, M.D. Modo de ação da peçonha da aranha armadeira, Phoneutria nigriventer (Keyserling, 1891), nas aurículas isoladas de cobaio. Ciênc. e Cult., 40:181-185, 1988. 7. VITAL BRAZIL & VELLARD, J. Contribuição ao estudo do veneno das aranhas II. Mem. Inst. Butantan, 2:3-77, 1926. 62 cm SciELO 10 11 12 13 14 15 16 Mem. Inst. Butantan, 52 (supl.), 1990 ACCIDENTS BY LOXOSCELES Francisco O. S. França,, Hospital Vital Brazil, Instituto Butan¬ tan, São Paulo. Spiders of the genus Loxoscetes belonging to the subfamily Loxoscelinae, fa- mily Scytodidae are distributed in America, Europe, Asia and África. In America there exist at least 50 species, seven of them in Brazil: L. gaúcho, L. laeta, L. intermedia, L. adeiaide, L. amazônica, L. similis 4 . Their body length is 1 to 1.5 cm, and with the legs extended up to 4 cm. The color varies from light brown to dark brown. The spider is commonly known as brown spider or violin spider 3 ' 4 . In 1989, 403 accidentes were probably caused by spiders of the genus Lox- osceles, according to data sent by 16 Brazilian States. At the Hospital Vital Brazil (I.B.) approximately 50 suspected cases of Loxoscelism are treated per year 2 ' 3 . The spider is shy causing accidentes only when crushed against the skin, as gener- ally occurs when the victim is dressing, or sleeping. Thus the most frequent sites of lesions are on the thigh, arm and torso. Seventy percent of the patients seek medicai help 24 hours after the accident, which coincides with the onset of the more florid symptoms. In at least 10% of the cases, the spider is recovered 2 . The clinicai response may vary from a slight local reaction to death. The syste- mic reaction is not necessarily proportional to the local reaction and vice-versa as the systemic symptoms may develop before any local reaction is noted. The evolution of the symptoms is related to the amount of injected venom, the locali- zation of the bite and the immunological condition of the patient. Loxoscelism may present itself clinically as a cutaneous form (97% of the cases) or as cutaneous-visceral form (3%) 2 . Up to now, routine tests for the diagnosis of Lox¬ oscelism do not exist 7 . The bite causes a slight transitory pain about 2 to 8 hours after the accident and pruritus as well as formication, pain and local erythema may arise. Afterwards a central ecchymosis appears and gradually expands. It is characterized by ir¬ regular margins, surrounded by an aureola of paleness and erythema, that in 90% of the cases evolves to a central necrosis 7 . This lesion called plaque, is of a vari- able extention and lies on an edematous and infiltered layer. Usually the lesion is painful and is not accompanied by enlargement of local lymph nodes 6 . 63 ff 'SciELO 0 11 12 13 14 15 16 1 Mem. Inst. Butantan, 52 (supl.), 1990 The appearance of vesicles and blisters with a serous or serous hemorrhagic con- tent and posterior ulceration is not uncommon. Cicatrization is slow, with a mum- mified central area, generally occurring within the first 4 weeks. The wound may develop a residual scab or cheloid. In 5% of the cases, mainly on the face, an edematous form occurs that is not necrotic being characterized by an extense phlogistic process. Some signs and/or systemic symptoms with fever, indisposi- tion, migraine, headache, nausea, vomiting, myalgia, exanthema may occur in variable percentage (25-75%) of the cases. The cutaneous-visceral form is characterized by the presence of clinically evi- dent hemolysis in addition to the cutaneous lesion and signs and systemic sym¬ ptoms described earlier. The onset of the hemolytic process occurs within the first two days in nearly all the cases. The patients develop hemolysis, hemolytic anemia, icterus, hemoglobinuria, hematúria, that may evolve with acute insuffi ciency, DIC, convulsion and coma. This syndrome does not have any connec- tion with sex and age of the patient, season, site and seriousness of the lesion 6 ' 7 . Specific serumtherapy using either antiloxoscelic serum (ALS) or antiarachnidic serum (AAS) is recommended for both forms of Loxoscelism. For the cutaneous forms, 5 ampoules of ALS or AAS are indicated, preferentially by the intravenous route. Several authors recommend also the use of DDS (diamine-diphenyl- sulphona) in an oral dosage of 50 to 200 mg/day. The use of antihistamines, corticoids and early surgical excision have been disputed 5 - 7 . In the case of residu¬ al lesions, surgical correction may be necessary. For the cutaneous-visceral form, 10 ampoules of LAS or AAS are indicated. The use of corticotherapy is recom¬ mended. Also, general rules of support are necessary, with special regard to the evaluation of the renal function and the hematologic parameters. REFERENCES 1. BRASIL. Ministério da Saúde. SNABS. Al. Aracnídlcos. Boletim n° 23. 1990. 2. CARDOSO, J. L. C. et alii. Loxoscelismo: estudo de 244 casos. In: CONGRESSO BRASILEIRO DE DERMATOLOGIA, 24, e JORNADA BRASILEIRA DE DERMATOLO¬ GIA SANITÁRIA, 1. Resumos. 1977. 3. CARDOSO, J. L. C.; FRANÇA, F.O.S.; VON EICKSTEDT, V.R.D.; BORGES, I.; NOGUEIRA, M. T. Loxoscelismo: estudo de 242 casos (1980-1984). Rev. Soc. bras. Toxicol., 7:58-60, 1988. 4. LUCAS, S. Spiders in Brazil. Toxicon, 26: 759-72, 1988. 5. REES, R.S. et alii. Brown recluse spider bite: a comparison of early surgical excision and Dapsone and delayed surgical excision. Ann. Surg., 202: 126-130, 1985. 6. SCHENONE, H., SAAVEDRA, T.; ROJAS, A.; VILLARROEL, F. Loxoscelismo en Chile: estúdios epidemiológicos, clínicos y experimentales. Rev. Inst. Med. Trop. São Paulo, 37:403-15, 1989. 7. WASSERMAN, E. J. et alii. Loxoscelism and necrotic arachnidism. J. Toxicol. Clin., 27:451-72, 1983/1984. 64 í, | SciELO Mem. Inst. Butantan, 52 (supl.), 1990 01. NOMENCLATURA EM TOXINOLOGIA: PEÇONHA OU VENENOS? José Carlos de Freitas, Instituto de Biociências, USP. São Paulo. Em todos os níveis da escala filogenética encontramos exemplos de ataque, defesa e outros com¬ portamentos baseados em substâncias repelentes, paralisantes ou de outras ações biológicas. Durante milhões de anos de evolução os organismos desenvolveram um refinamento destas subs¬ tâncias para a captura de presas e defesa química e a nomenclatura adotada em toxinologia leva em conta esses aspectos. RUSSELL (1967, 1971) enfatizou que animais venenosos ou peço¬ nhentos são encontrados em cada filo, fazendo exceção às Aves e as propriedades das toxinas de peçonhas diferem muito das toxinas presentes nos venenos, mesmo em representantes de um mesmo filo. F.E. RUSSELL (comunicação pessoal) ainda considerou o termo biotoxina um pleonasmo, uma vez que o termo toxina já denota sua origem biológica. VITAL-BRAZIL (1973) empregou os termos peçonhas e venenos considerando as propriedades farmacológicas das to¬ xinas integrantes. Nesta mesma data FREYVOGEL & PERRET conceituaram venenos (do inglês "poisons"), produtos metabólicos produzidos ou estocados em órgãos, que em condições na¬ turais afetam por ingestão e artificialmente podem atuar pela via parenteral. As peçonhas (do inglês "Venoms") originam-se em glândulas especializadas dotadas de dutos secretores asso¬ ciadas ou não a estruturas inoculadoras, atuam pela via parenteral e podem ser destruídas quan¬ do ingeridas. 02. CROTOXIN, THE NEUROTOXIN FROM THE VENOM OF CROTALUS DURISSUS CASCAVELLA. Barbara Exceli Hawgood, Dep. Farmacologia, Unicamp, Campinas S.P. Crotoxin is a complex of 2 polypeptide subunits (i) a basic phospholipase A2 and (ii) a modified phospholipase A 2 that lacks enzymic activity. The subunits can be readily separated but must combine for high neurotoxic potency (Bon et ai, 1989). Crotoxin produces respiratory paralysis primarily by blocking the release of transmitter from motor nerve terminais in the diaphragm. Unlike botulinum toxin, it does not cross the membrane to act internally (Trivedi et ai,, 1989). Crotoxin produces a complex series of changes in both spontaneous and nerve-evoked transmit¬ ter release prior to complete blockade of neurotransmission. Although phospholipase A 2 activity is involved in the neurotoxic action, there is no evidence that either production of prostaglandins (Edwards et. ai,, 1990) or activation of protein kinase C (Rodrigues-Simioni et al, 1990) is en- volved in any of the stages. It has been proposed that crotoxin acts by producing localized hydrolysis of membrane phospholipids at transmitter release sites in the nerve terminal (Hawgood, 1989). In experimental animais, crotoxin can also produce focal lesions in skeletal muscle, kidneys and lung. Bon, C., Bouchier, C., Choumet, V., Faure, G., Jiang, M., Lambezat, M., Radvanyi, F., Saliou, B. (1989) Acta Physiol. Pharmac. Lat. Americ. 39, 439. Edwards, J., Hawgood, B.J., Smith, I.C.H. (1990) Toxicon in press. Hawgood (Exceli), B.J. (1989) Acta Physiol. Pharmac. Lat. Amer. 39, 397. Rodrigues-Simioni, L., Hawgood, B.J., Smith, I.C.H. (1990) Toxicon in press. Trivedi, S., Kaiser, L, Tanaka, M., Simpson, L. (1989), J. Pharmac. exp. Ther. 251, 490. 03. ESTUDO DA COAGULAÇÃO EM ACIDENTE CROTÁLICO EM REGIÃO DE C. d. cascavella. Hui 1 N. Fan (1); José Ft.F. Oliveira (2); Francisco O.S. França (1 ); Sandra C. B. Castro (3); João L. C. Cardoso (1); Aura S. Kamiguti (3). (1) Hospital Vital Brazil e (3) Seção de Hematologia do Inst. Butantan, S. Paulo, SP. (2) Hospital Giselda Trigueiro da Universidade do Rio Grande do Norte, Natal, RN. Dados sobre acidente crotálico no Brasil têm sido referidos para regiões de distribuição de C. d. terrificuse C. d. collilineatus, correspondendo às regiões sul, sudeste e centro oeste do Brasil. No presente estudo são reportados dois acidentes humanos com diagnóstico clínico de acidente crotálico, ocorridos no Rio Grande do Norte, área de distribuição geográfica de C. d. cascavella. Do ponto de vista clínico-laboratorial, foram observados nos dois pacientes fenômenos neuro- paralíticos (ptose palpebral, oftalmoplegia, midríase, sialorréia), miotóxicos (míalgia generaliza¬ da, urina de cor escura), e sangue incoagulável, quando na admissão hospitalar. Ambos foram classificados como acidentes moderados, sendo efetuados os seguintes testes la¬ boratoriais: tempo de protombina; tempo de tromboplastina parcial ativado; dosagem de fibrino- gênio e dos produtos de degradação do fibrinogênio (e fibrina); teste de d-dímero e de mioglobina monitorados de 6 em 6 horas até a alta dos pacientes. Foram submetidos à terapêutica com antiveneno específico, cuja evolução ocorreu sem complicações e com normalização das altera¬ ções laboratoriais em 24 horas. Esta apresentação tem por objetivo fornecer subsídios ao me¬ lhor conhecimento dos aspectos regionais do ofidismo no Brasil. 67 cm 2 3 L. 5 6 11 12 13 14 15 16 Mem. Inst. Butantan, 52 (supl.), 1990 04. ESTUDO DE VARIAÇÕES CONFORMACIONAIS DA CROTOXINA, FOSFOLIPASE E CROTAPOTI- NA EM SOLUÇÃO POR SAXS. José Ramon Beltranl 1); Carlos Julio Laurel 2). (1) Departamento de Física — IBILCE — UNESP — São José do Rio Preto. (2) Faculdade de Medicina de Ribeirão Preto. Mediante a técnica de espalhamento de raios X a baixo ângulo, estudaram-se a forma e tama¬ nho do complexo crotoxina, proteína neurotóxica extraída do veneno da Crotalus durissus terrifi- cus, em diferentes condições de concentração (60, 50, 40, 30 e 20 mg/ml), pH 1,5 e temperatura de 24°C, visando a determinação dos seguintes parâmetros estruturais: raio de giro Rg, volume hidratado V, área da superfície da proteína S e máxima dimensão D da molécula. O estudo quali¬ tativo das curvas da função/distribuição de distâncias P(r) confirma claramente a presença no complexo crotoxina de duas proteínas associadas (crotapotina e fosfolipase) se as comparar- -mos com as funções P(r) calculadas usando modelos de agregados diméricos para elipsóides de revolução prolatos (O. Glatter, J. Appl. Cryst., 12, 166, 1979). Um estudo similar das curvas de espalhamento e as funções P(r) foi feito para a crotapotina e fosfolipase com o objetivo de estabelecer o tipo de enlace dimérico no complexo crotoxina. 05. BIODISTRIBUIÇÃO DE CROTOXINA NATIVA E IRRADIADA EM ÓRGÃOS DE CAMUNDONGOS USANDO COMO TRAÇADOR 0 I. Nanei do Nascimento (1); Miriam C. Guarnieri Cruz (1) Ulysses Tomac Jr. (1); Heitor F. de Andrade Jr. (2); José Roberto Rogero (1). (1) Div. de Radiobiologia, IPEN, São Paulo, SP: (2) Lab. de Protozoologia, IMT, USP, São Paulo, SP. A crotoxina, por ser o principal componente do veneno crotálico, tem sido alvo de estudos que visam elucidar seus aspectos bioquímicos e farmacológicos. O presente trabalho tem como ob¬ jetivo comparar a biodistribuição da crotoxina nativa e irradiada (2000 Gy) em órgãos de camun¬ dongos, dado que essa proteína quando submetida à radiação ionizante tem sua toxicidade reduzida preservando no entanto sua propriedade antigênica. Para esse ensaio, as amostras foram mar¬ cadas com 125 l e inoculadas separadamente em grupos de 10 animais í-30g) por via endove¬ nosa. Os animais foram sacrificados após 0,25; 1; 2; 3; 5 e 24 horas, sendo retirados os seus órgãos para posterior contagem em um contador Nuclear Chicago. A presença de crotoxina irra¬ diada ou não foi dada relacionando-se as contagens por minuto com o peso do órgão (mg). Os resultados mostraram um acúmulo significativo da proteína marcada no fígado, baço, músculo e cérebro com pico na terceira hora tanto para crotoxina nativa quanto para a irradiada. Numa análise inicial dos dados obtidos para o rim podemos verificar que ocorre uma concentração de crotoxina nativa nas primeiras 3 horas nesse órgão, o que não é observado com a crotoxina sub¬ metida à radiação. Sendo assim, aparentemente o rim funciona somente como órgão de elimi¬ nação no caso da crotoxina irradiada. 06. DISTRIBUIÇÃO GEOGRÁFICA DAS SERPENTES CROTALUS DURISSUS VARIEDADE CROTAMI- NA POSITIVO NOS ESTADOS DE MATO GROSSO E MATO GROSSO DO SUL. Silveira U.S. (1); Diniz M.R.V. (2); Santos, S.M. (3). (1) Departamento de Toxicologia e Farmacologia MS; (2,3) Fundação Ezequiel Dias MG. Introdução — Schenberg (1959) determinou a distribuição geográfica das serpentes Crotalus durissus v ariedade crotamina positivo para o Estado de São Paulo, Diniz et al. (1988) determina¬ ram esta distribuição para o Estado de Minas Gerais. Pretendemos concluir verificando até que paralelo é possível encontrar serpentes crotalus com esta propriedade. Objetivo — Conhecer a distribuição de populações de serpentes Crotalus durissus variedade cro¬ tamina positivo a oeste dos Estados de São Paulo e Minas Gerais. Materiais e Métodos — Foram testados venenos de serpentes Crotalus durissus coletados indivi¬ dualmente. Aplicou-se 0,5 mg de veneno em cada camundongo Mus musculus com peso entre 18 a 22 g, via subcutânea e aguardou-se 30 min. para surgimento do sintoma de paralisia dos membros traseiros. Foram utilizados 2 camundongos para cada veneno testado. Resultados — Os mapas demonstram a distribuição dos dois Estados. Mato Grosso Conclusões — Verificou-se que as populações variedades crotamina positivo se encontram pró¬ ximas ao Rio Paraná e ao Sul do Estado de Mato Grosso do Sul. Para Oeste e Norte do Estado e no Estado de Mato Grosso a Crotamina praticamente não é encontrada. 68 cm SciELO 10 11 12 13 14 15 16 Mem. Inst. Butantan, 52 (supl.), 1990 07. MICRURUS NIGROCINCTUS VENOM INDUCED EFFECTS ON ISOLATED MOUSE PHRENIC NERVE DIAPHRAGM AND CHICK BIVENTER CERVICIS PREPARATIONS. Léa Rodrigues Simioni 1 , José C. Cogo\ José Maria Gutierrez, 2 , Júlia Prado Franceschi^. Departamento de Farmacologia, FCM/UNICAMP — Campinas (SP) 1 . Instituto Clodomiro Picado, Universidade Costa Rica, Costa Rica 2 . The effect of Micrurus nigrocinctus venom was studied in the isolated mouse phrenic nerve di- aphragm and chick biventer cervicis preparations. A dose-dependent (5,10,20 and 40 ug/ml) blockade was observed in the mouse phrenic nerve diaphragm in response to direct or indirect stimulation. The addition of neostigmine (5-10 ug/ml) induced partial and rapid reversal followed by Progressive blockade. In the biventer cervicis, the blockade and contracture evoked by the venom were more intense and faster than in the mouse phrenic nerve diaphragm preparation and it was not reversed by several washings or by neostigmine. The response of curarised prepa¬ rations, directed stimulated, were blocked by the venom. In the mouse diaphragm the venom was able to diminish both frequency (within 15 minutes) and amplitude (after 45 minutes) of the m.e.p.p.s. as well as the resting potential membrane. These results suggest that M. nigrocinctus venom exerts a presynaptic action. In addition, creatinine kinase release was generally increased by 9 fold above pre-venom values in both preparations according to the myonecrosis previously described (Gutierrez et al., 1980). Gutierrez, J.M.; Chaves, F.; Rojas, E. and Bolanos, R. Toxi- con 18: 633-639 1980. 08. PHILODRYAS OLFERSII: MORPHOLOGICAL, HISTOCHEMICAL STUDIES OF DUVERNOVS GLAND. VENOM EXTRACTION. M. Graça Salomão ", G. Puorto', Fátima Furtado", Sawaya, P .''' "Seção de Flerpetologia, * "Seção de Venenos — Instituto Butantan, """Depto. Fisiol. IBUSP. Envenomations by the snake Philodryas olfersii cause serious local effects, mainly hemorrhage and edema. P. olfersii, a Colubridae-Xenodontinae is an opisthoglyphous snake (Lichtenstein, 1823) with a well developed Duvernoy's gland connected with a grooved tooth. These glands are located in the postocular region, behind the supralabial glands. Comparing the size of the Duvernoy's gland of P. olfersii to Sibynomorphus mikani (aglyphous colubrid snake), Bothrops jararaca and Micrurus frontalis (venomous viperid and elapid snakes), was found the value of 15x7 mm for P olfersii, 20 x 10 mm for B. jararaca and M. frontalis and only 1.0 x 1.5 mm for S. mikani. The glandular duct is central in P. olfersii and S. mikani, resulting from the con- fluence of the acinous ducts, while in the venomous snakes the tubules join to form an anterior ejector canal. Flistological studies of Duvernoy's gland showed a predominance of serous cells in. P. olfersii (80%). Flowever, the mucous cells (70%) predominated in S. mikani. While in B. jararaca and M. frontalis mucous cells are found only in the accessories glands. The ultra-structure of Duvernoy’s gland cells showed strong eletro-dense secretion granules in P. olfersii, similar to those of B. jararaca and M. frontalis. Flistochemical analysis showed also differences in the polysaccharides and amino-acids contents of the Duvernoy's gland cells. While in P. olfersii was found only neutral polysaccharides, in S. mikani were present neutral, acidic and sulphated ones. No stain was obtained in B. jararaca, and M. frontalis. The detection of amino-acids showed the presence of arginine, tryptophan, hystidine and lysine in all examined glands. Tyrosine was absent only in B. jararaca. Considering the position of the fangs at the end of the maxilla in P. olfersii, which makes difficult to extract the venom, milking was achieved by introducing the fangs into 100 /xl micropipettes. As for the venom from S. mikani, this was obtained after trituration of the removed Duvernoy's glands. When compared to the venom of. B. jararaca, the protein content of P. olfersii is 75%, while of the S. mikani only 7.5%. The proteolytic activity of the venom of P. olfersii was 208% more than B. jararaca, and S. mikani was 16% from that. Doses of 45 ug i.p. in mice (18-22g) killed more than 50%. 09. BIOLOGICAL AND BIOCHEMICAL CHARACTERIZATION OF THE VENOM OF PHILODRYAS OL- FERSII. Marina T. Assakura (1); Maria da Graça Salomão (2); Giuseppe Puorto (2); Fajga R. Man- delbaum (1). (1) Serviço de Bioquímica e (2) Secção de Herpetologia, Instituto Butantan, São Paulo, SP. Accidents with the opístoglyphous colubrid snake Philodryas olfersii âre rare. However, enveno¬ mations are very serious. The local symptoms are extensive hemorrhage and edema. Analysis of venom by polyacrylamide gel electrophoresis (PAGE) showed a major band and a great num- ber of protein bands with low concentrations. Biological and biochemical studies were performed with venom. Hemorrhagic activity is very strong in this venom, the minimum hemorrhagic dose (MHD) to produce a spot of 1 cm 2 was 0.5 /xg, as tested by intradermal injection in mice. The hemorrhagic principie of this venom is antigenically different from the hemorrhagic principies of the Viperinae and Crotalinae subfamilies. This venom did not cross-react with antibodies to the hemorrhagic factors isolated from the venoms of B. neuwiedi and B. jararaca. Also the venom was not neutralized by these antisera. It has no thrombin-like activity, as no clotting was ob- 69 cm 2 3 L. 5 6 11 12 13 14 15 16 Mem. Inst. Butantan, B2 (supl.), 1990 served in horse plasma or human fibrinogen. Plasma or fibrinogen previously incubated with this venom prolonged the clotting time of thrombín or bathroxobin. The clotting time increased with the increasing of the incubation time of plasma or fibrinogen with the venom of P. olfersii. Hu¬ man fibrinogen incubated with the venom at the proportion 20:1 (w/w) showed in SDS-PAGE the destruction of A a and B /3-chains after 3 min. The y-fibrogen Chain remained unchanged even after 2 h of hydrolysis. The enzyme has also a high collagenolytic activity. Collagen type I was incubated with the venom at the proportion 20:1. Hydrolysis of all chains was observed already in 30 min and total destruction of the collagen was achieved at 24 h. 10. CARACTERIZAÇÃO EDEMATOGÊNICA DE VENENOS CROTALÍNEOS DA AMÉRICA DO SUL. An¬ drade. S.fí.M.; Martins, M.M.; Sanchez, E.F. (FUNED-MG); Diniz, C.Ft. e Ferreira-Alves, D.L. (ICB-UFMG, Belo Horizonte-MG). Embora o edema seja o indicador da severidade de muitos acidentes com viperídeos e mesmo de alguns elapídeos, o parâmetro foi desconsiderado na caracterização toxicológica laboratorial proposta por Theakston e Reid (Buli. W.H.O. 61(6): 949-956, 1983). Camundongos albinos (Swiss, 18-22 g, FUNED) foram injetados por via intraplantar com 50 p\ de solução de veneno ou 50 /d de salina, a pata contralateral permanecendo como controle - a resposta expressa com A % do peso das patas. As curvas tempo-resposta indicaram A % máximo de 15 a 20 minutos, exce¬ to B. moojeni, com 30 minutos e as curvas dose-resposta forneceram a ED50 e edema máximo. ESPÉCIE ed 50 g A % Máx. ESPÉCIE ed 50 % Máx. B. jararaca-MG 18,4 71 B. castenaldi-PA 26,0 61 B. jararaca-FUNED 21,1 67 B. jararacussu-MG 9,2 83 Bra - Bot 001 22,6 77 B. leveurus-BA 10,6 72 B. alternatus-RGS 24,2 68 B. moojeni-MG 7,0 88 B. alternatus-Arg. 45,2 77 B. altiplans 22,6 71 B. alternatus L3 13,1 64 B. asper 7,0 79 B. atrox-Peru 30,0 71 C. d. terrificus-MG 32,0 56 B. atrox-PA 39,4 63 C. d. terrificus-Bol. 36,8 53 B. atrox L 24 8,0 75 C. d. collineatrus-GO 9,2 58 B. newiedii-Arg. 13,0 71 L. m. muta-Peru 15,0 76 B. newiedii II 15,0 78 L. m. muta-PA 68 Apoio financeiro: FUNED-CNP q -FAPEMIG 11. EDEMA E PERMEABILIDADE VASCULAR INDUZIDOS PELO VENENO DA BOTHROPS NEUWIE- Di PAULOENSIS: Ronilson A. Moreno; José E. Zambelli e Júlia Prado-Franceschi. Departamento de Farmacologia, Faculdade de Ciências Médicas, UNICAMP Campinas, São Paulo. Acidentes botrópicos caracterizam-se por induzir profundas alterações locais traduzidas por vo¬ lumoso edema, hemorragia e necrose do tecido atingido (Vital Brazil, 1982). Na tentativa de relacionar a atividade edematogênica com alterações na permeabilidade vascular, camundon¬ gos Swiss, machos (20g + 2) foram submetidos a injeção subplantar da peçonha de Bothrops neuwiedi pauloensis na pata posterior (doses de 0,1 a 2,7 ^g/pata), sendo os animais previa¬ mente tratados com azul de Evans (25 mg/kg). 0 edema foi avaliado pelo aumento do peso da pata, em diferentes intervalos de tempo, nos quais também foi extraído o conteúdo de corante em formamida (48 h em banho-maria 37°C). A concentração de azul de Evans foi então deter¬ minada por espectrofotometria (619 nm), comparando-se os valores obtidos com os da curva padrão. Tanto edema quanto permeabilidade vascular mostraram-se aumentados com maior evi¬ dência entre os tempos de 30 a 60 minutos. A análise estatística dos dados mostrou forte corre¬ lação entre os resultados obtidos para os dois eventos. 12. ESTUDO DA ATIVIDADE MIOTÓXICA DE VENENOS BOTRÓPICOS. Ana M. Moura-da-Silva { 1); Diva F. Cardoso (1) e Martha M. Tanizaki (2). (1) Centro de Pesq. e Form. em Imunologia e (2) Serviço de Bioquímica, Instituto Butantan, São Paulo-SP. Demonstramos recentemente a existência de antígenos comuns nos venenos botrópicos exceto para uma banda localizada entre 14 e 18 kDa que se encontra presente apenas em 4 dos 9 ve¬ nenos analisados. Do veneno de B. jararacussu foi isolado por gel filtração em Sephadex G-75 e SP-Sephadex C-25, um componente de baixo peso molecular e que apresenta intensa ativida¬ de miotóxica. Anticorpos preparados contra esse componente purificado reconhecem apenas uma banda de cerca de 18 kDa nos venenos de B. jararacussu, B. moojeni, B. neuwiedi e B. pradoi fracionados em SDS-PAGE. A atividade miotóxica do veneno de B. jararacussu é grande¬ mente reduzida pela prévia incubação com os anticorpos obtidos contra o componente purifica¬ do, assim como pelos soros antivenenos miotoxina + . Os venenos que não apresentam o 70 cm SciELO 10 11 12 13 14 15 16 Mem. Inst. Butantan, 52 (supl.), 1990 componente restrito (miotoxina -) mostram uma baixa atividade miotóxica que não é reduzida pela prévia incubação com anticorpos antimiotoxina. A letalidade do veneno de B. jararacussu é reduzida pelos anticorpos antimiotoxina e pelos soros antivenenos miotoxina + e miotoxina Porém os anticorpos antimiotoxina não são capazes de reduzir a letalidade do veneno de B. cotiara (miotoxina -i. Os dados apresentados sugerem que a miotoxina é restrita a alguns vene¬ nos botrópicos; que ela participa na letalidade destes venenos e que os anticorpos específicos são importantes para sua neutralização. Auxílio CNPq (402.380/89-4/BM/FV/PQ) 13. ESTUDO COMPARATIVO DO EFEITO NEUTRALIZANTE DOS SOROS ANTIBOTRÓPICO (SAB) E ANTIBOTRÓPICO/CROTÁLICO (SAB-C) SOBRE A ATIVIDADE MIOTÓXICA DO VENENO DE Both- rops jararacussu (Bjssu) EM CAMUNDONGOS. M. Cristina dos Santos (1); Luís R. C. Gonçalves (2); Consuelo L. Fortes Dias (4); Yara Cury (2); José Maria Gutierrez (5); M. Fátima D. Furtado (3). (1) Dept°. lmunologia,ICB-USP, (2) Seção de Fisiopatologia Experimental e (3) Seção de Vene¬ nos do Instituto Butantan, S. Paulo-SP; (4) Fundação Ezequiel Dias, Belo Horizonte—MG; (5) Instituto Clodomiro Picado, Costa Rica. A mionecrose é um dos efeitos causados pelo veneno de Bjssu. Deste veneno, foi isolada uma miotoxina com homologia estrutural à fosfolipase A2 (PLA2i, mas sem atividade enzimática. O veneno de Crotaius durissus terrificus (Cdt) apresenta uma atividade miotóxica que vem sendo atribuída à PLA2, o componente básico do complexo orotoxina. Camundongos imunizados com PLA2, isolada desse complexo, tornaram-se resistentes a ação letal e miotóxica do veneno de Cdt. Pela técnica de W. Blotting, o veneno de Bjssu apresentou três proteínas com identidade imunológica à PLA2 da crotoxina. Com base nestes dados, foi comparada a eficiência dos soros comerciais produzidos pelo Instituto Butantan, o SAB e o SAB-C, na neutralização do veneno de Bjssu. A neutralização do veneno foi feita "in vitro", incubando-se, por 30 min. a 37° C, doses fixas de veneno com diferentes concentrações de antivenenos. As misturas foram injeta¬ das, pela via intraperitoneal para avaliação da letalidade, e pela via intramuscular para a avalia¬ ção da mionecrose (dosagem de CK séricol e hemorragia (dosagem da hemoglobina no tecido muscular). 0 SAB e o SAB-C apresentaram a mesma eficiência em neutralizar a ação hemorrági¬ ca (ED50 28.5 e 27/4 av/mg ven.). Para a ação letal, o SAB apresentou uma potência de 2.4mg/ml, enquanto que o SAB-C, uma potência de 3.45mg/ml e para a ação miotóxica, o SAB e o SAB-C apresentaram, respectivamente, ED50 de 560 e 140/4 av/mg ven., demonstrando que o SAB-C possui uma capacidade sensivelmente maior em neutralizar essas atividades. Esses dados suge¬ rem que o tratamento com o SAB-C poderia ser vantajoso nos casos de acidentes causados por B. jararacussu. 14. LESÃO MUSCULAR PRODUZIDA POR VENENO DE ABELHAS AFRICANIZADAS (VAA) EM RA¬ TOS. Azevedo-Marques, M.M. (V, Ferreira, D.B. (2) e Costa, R. S. (2). (1) Dept° de Clínica Médica e (2) Patologia da Faculdade de Medicina de Ribeirão Preto-USP. Neste trabalho são descritas as alterações anátomo-patológicas observadas à microscopia ópti¬ ca em diferentes tipos de tecido muscular sob a ação de VAA. Foram inoculados 80 ratos machos Wistar com 1,5 /4/100 g de peso de VAA por 4 vias. Colhido sangue por punção cardíaca, sob anestesia, até o sacrifício do anima' pós 4, 7 e 24 h da ino¬ culação, nos injetados por via subcutânea (SC), intramuscular (IM) e intraperitoneal (IP) e aos 2 minutos nos injetados por via endovenosa (EV). Os 80 ratos do grupo controle receberam igual volume, pelas mesmas vias, de solução 0,15M de salina. As enzimas séricas creatinoquínase (CK) e aspartato aminotransferase (AST) foram quantificadas, em U/l, em pool de soro de 8 ani¬ mais, usando teste U/V optimizado. Os fragmentos de musculo cardíaco e esquelético colhidos no local e à distância, colocados em formol tamponado a 10%, incluídos em parafina, cortados em espessura de 4/i, foram analisados em lâminas coradas pela hematoxilina-eosina (HE) e fos- fotungstica (PTAH). 1) Valores das enzimas séricas em U/L ( ) grupos controle. IM IP SC EV (2 minutos) X AST 4 h CK 476 ( 75) 1490 (423) 1740 ( 75) 20060 (334) 200 (120) 520 (468) 440 ( 85) 1940 (386) 487 ( 72) 220 (149) 1440 ( 75) 8460 (357) 140 ( 75) 423 (334) 391 ( 68) 149 (125) 520 ( 52) 453 (190) 140 ( 75) 423 (371) (2) Microscopia: alterações observadas nos 3 horários. Via SC - necrose lítica adiposo-muscular, infiltrado inflamatório agudo (iia); IM - edema, hemorragia, iia, mionecrose-miocitólise (m-m); 71 cm iSciELO 0 11 12 13 14 15 16 Mem. Inst. Butantan, 52 (supl.), 1990 IP - iia,m-m da parede abdominal. Nestas 3 vias não foram encontradas lesões musculares à distância. Via EV - iia, m-m em áreas irregulares em diversos músculos. Não houve lesões mio- cárdicas em nenhum dos grupos. Conclusões: II O VAÁ apresenta atividade miotóxica local e sistêmica, dependendo da via de inoculação; 2) As enzimas séricas traduzem a intensidade das lesões musculares de acordo com a via utilizada. Em ordem decrescente de valores IP, EV, IM e SC. 15. ESTUDO DA ATIVIDADE MIOTÓXICA DA BOTHROPSTOXINA. P.A. Me/od); M. I. Hornsi- Bran¬ deburgo (2); J. R. Giglio (3); G. Suarez-Kurtz I 1). (11 Dep. de Farmacologia, ICB, UFRJ; (2) Dep. Geociências, Inst. Biociências, UNESP, S. J. Rio Preto, SP; (3) Dep. Bioquímica, USP, R. Preto, SP. O veneno de B. jararacussu é veneno crotalídeo que tem sido estudado em nosso meio e dele foram extraídos vários componentes com atividade miotóxica. O objetivo de nosso trabalho foi de testar em nossos modelos experimentais a atividade miotóxica de uma proteína isolada do veneno de B. jararacussu por Homsi-Brandeburgo et al. (Toxicon 26, 1988) denominada Both- ropstoxina (BthTxl. Nos experimentos "in vitro" usamos o músculo extensor digitorum longus de camundongos banhados em meio nutridor e medimos o efluxo da enzima sarcoplasmática creatinofosfoquinase (CK). Este meio era renovado a cada 40 min, quando passamos então a acrescentar as substâncias em estudo. A BthTx (25 ^g/ml) aumentou o efluxo basal de CK de 0,26 + 0,03 U.g^h ' 1 (n = 181 para 7,18 + 2,54 U.g^h -1 (n = 9) após 160 min de exposi¬ ção. Ao acrescentarmos simultaneamente a BthTx heparina (50^g/ml, n = 41 ou Wedelolacto- na (10 /rg/ml, n = 4), a atividade miotóxica da BthTx foi completamente bloqueada. Nos experimentos ''in vivo" camundongos receberam injeção I.M. de BthTx (1 /rg/g) e medimos o aumento da atividade de CK no plasma após a injeção. Os animais pré-tratados com Wedelolac- tona (1 nglçj) previamente ou associado a BthTx tiveram reduzidos em cerca de 40% a atividade de CK no plasma. Heparina 5 /xg/g em mistura com a BthTx I.M. bloqueou a atividade miotóxica da BthTx. Apoio: CNPq, FINEP, CEPG-UFRJ 16. PLATELET AGGREGATION AND HYDROLYSIS OF COLLAGEN, FIBRONECTIN, FIBRIN AND Fl- BRINOGEN BY TWO BASIC SERINE PROTEINASES ISOLATED FROM BOTHROPS MOOJENI VENOM. Solange M.T. Serrano (1); Maria F.C. Matos! 2); Misako U. Sampaio (2); Cláudio A.M. Sampaio (2); Fajga R. Mandelbaum (1). (1) Serviço de Bioquímica, Instituto Butantan e (2) Departamento de Bioquímica, Escola Paulista de Medicina. The local effects and the disturbs in blood coagulation observed in the envenomation by Bothrops moojeni are attributed to various proteinases present in this venom. It presents a high content of basic components. Two proteinases with distinct specificities were purified from the basic fraction by gel-filtration on Sephadex G-100, followed by adsorption of the acidic components to DEAE- Sephacel (pH 7.5). Isolation was achieved by ion-exchange chromatography on SP-Sephadex C-50 (pH 7.5). They are free of hemorrhagic, blood-clotting and phospholipase A 2 activities, but still present 15% of the caseinolytic activity present in the crude venom. Both proteinases hydro- lyze natural substrates such as fibrinogen, fibronectin and type I collagen.Proteinase I is more active on collagen than proteinase II. Proteinase I hydrolyzes the 7 , 0, a 1 (I) and a 2 (I) chains of the collagen giving a great number of peptides of M.W. ranging from 80,000 to 10,000. On the other hand proteinase II hydrolyzes only 7 and (3 chains very, slowly, producing peptides of high molecular weight (100,000 to 80,000). Fibrinogen and fibronectin were similarly degraded by both enzymes. The two proteinases are distinguished by the following properties: A — Proteinase I not only causes platelet aggregation at a concentration of 10 ' 8 M but also en- hances the ADP aggregating activity. It does not hydrolyze fibrin and has low activity on TAME (26 ftM/min/mg). B — Proteinase II hydrolyzes fibrin and has high activity on TAME (144 íiM/min/mg). 17. EFEITO DO VENENO DE Bothrops erythromeias NA AGREGAÇÃO PLAQUETÁRIA. Aldete Zap- pellini e Júiia Prado-Franceschi. Departamento de Farmacologia, Faculdade de Ciências Médicas, UNICAMP Campinas, São Paulo. Investigou-se 0 efeito do veneno de B. erythromeias na agregação de plaquetas humanas lava¬ das induzida por trombina (2,9 U/ml). O emprego de plaquetas lavadas justifica-se pela elevada atividade coagulante deste veneno em plasma. Sangue de voluntários adultos e sadios, que se abstiveram de drogas antiinflamatórias não esteroidais por pelo menos uma semana, foi coleta¬ do em citrato de sódio 3,8% (9:1). O plasma rico em plaquetas foi centrifugado (2700 rpm por 12 min.) e o pellet, ressuspendido em solução de Tyrode sem cálcio, na presença de lloprost (300 ng/ml). Após recentrifugação, o pellet foi ressuspendido em solução de Tyrode com cálcio (2,5 mMI. Verificou-se que o veneno, nas doses utilizadas (5 a 80 /xg/ml), inibiu a agregação plaquetária de forma dose-dependente. Considerando-se a hipótese de que fosfolipases presen- 72 cm SciELO 10 11 12 13 14 15 16 Mem. Inst. Butantan, 52 (supl.), 1990 tes nos venenos botrópicos parecem inibir o efeito agregante (Zingali et al., 1988), pesquisou-se ainda a correlação entre as atividades fosfolipásica e antiagregante nas frações obtidas em Se- phadex G-75. A possibilidade de que proteases presentes no veneno atuem degradando a trom- bina também foi examinada. 18. PADRONIZAÇÃO DOS MÉTODOS DE ESTUDOS DA ATIVIDADE COAGULANTE DE VENENOS IVEN.) OFÍDICOS EM ANIMAIS DE LABORATÓRIO. Sadala, L. - Fundação Ezequiel Dias IFU- NED); Diniz, C. R.& Ferreira-Alves, D.L. — ICB-UFMG, Belo Horizonte. No ven. das viperldeas encontram-se enzimas "thrombin-like'', que transformam fibrinogênio em fibrina e procoagulantes, que ativam fator X. Visamos padronizar este estudo em plasma de ratos (P) e camundongos, nos quais se determinam capacidade letal, hemorrágica, necro- sante, miotóxica, defibrinogenante, etc. dos ven. ofidícos. A 37°C, tubos de hemólise recebe¬ ram soluções na ordem: substrato (S), fibrinogênio humano (F) 6 % em salina (sal.) 0,15 M ou P em citrato (cit) 4% ou cit + heparina (cit. hep) 4 U/ml; sal.; ven. C. d. terrificus (VC) e 6 . jararaca (VB) — FUNED; cálcio (Ca) em tampão Tris-HCI 0,04M, pH 8,0 ou água. 0 tempo de coagulação (TC, procedimento padrão) mínimo foi de 15 seg. Expressou-se a velocidade relati¬ va de coagulação: VR = 15/TC. Para 100 /xl F + 50 /d VC ou VB (125 /ig/ml sal. 0,15M) + 50 /iI sal. 0,15 M + 50 /rl Ca 0,01 - 0.20 M em tampão, a curva de ativação pelo Ca foi em V invertido e VR atingiu max. em Ca 0,05 M seguida de inibição pelo aumento da força iônica (Fl) em alto Ca. Já em meios de menor Fl: 200 /il P + 50 /al VC (500 g/ml) ou VB (250 /ag/ml sal. 0,15 M) + 100 /tl Ca 0,01 - 0,20 M em água, a curva exibe platô em Ca 0,01 - 0,075 M para VC e 0,037 - 0,1 M para VB, seguido de inibição. Quanto à influência da Fl em 200 /al P + 50 /al VC (500 /tg/ml) ou VB (250/ag/ml sal. 0,03 M) + 100 /al Ca 0,05 M em água + 100 /al sal. 0,0047 - 0,61 M, vimos que VR aumenta inversamente à salinidade. Nas curvas de tS] : 10-200 /al P + 0 - 90/al sal. 0,02 M + 100 /al Ca 0,025 M em água + VC (500/ag/ml) ou VB (125 /ag/ml sal. 0,03 M). VR foi max. entre 20 - 50 /al, ocorrendo aparente inibição por excesso de P. Para P - cit, a VR para VB foi maior que a do P - cit. hep. diferença não observada para VC. Conclui-se que processo de colheita do P, Fl, [S] , Ca e outras variáveis devem ser controladas, e não apenas [ven.] como preconiza Theakston & Reid (Bulletin of the World Health Organization, 61(6), 1983). Apoio financeiro: CNPq, FUNED, UFMG. 19. BIOCHEMICAL AND BIOLOGICAL DIFFERENTIATION OF VENOMS FROM BOTHROPS SPECIES ( ATROX, ASPER, MARAJOENSIS AND MOOJENI). Marina T. Assakura (1): Maria F. D. Furtado (2); Fajga R. Mandelbaum (1). (1) Serviço de Bioquímica e (2) Seção de Venenos, Instituto Butantan, São Paulo, SP. The snake Bothrops atrox is found in regions along the Central America to south of Brazil. Until now there is no definitive zoological classification. Hoge (1965) based in the geographical distri- bution and morphological differences divided this speoies in B. asper, B. atrox, B. pradoi, B. marajoensis and B. moojeni. The venoms of these species compared to other Bothrops have a high content of proteolytic enzymes with very specific activities, such as, thrombin-like and pro-coagulant enzymes. To define the biological and biochemical differences inter and intra-species, venoms were collected from different localities. PAGE showed that there is no similarity of the protein distribution inter and intra-species, except 8 . moojeni which presented a similar compo- sitions of the proteins with low mobility. Proteolytic activity on casein of these venoms are very high but the variation is small, being B. atrox (Tucurui) the most active and 8 . asper (Atlantic coast) the lowest one. However, the proteases were differently distributed intra and inter-species, except in 8. moojeni the venoms showed similar patterns. Hemorrhagic activity of these venoms are very low. The minimum hemorrhagic dose (MHD) in mice of 8 . atrox varied from 4-6 g, 8 . moojeni from 3-8 /rg and 8 . asper from 3-5.5 /rg. Also the letality is low, LD 50 in mice varied 90-145 ng B. moojeni), 70-110 /tg (B. atrox) and 70-78 ng (B. asper). The clotting activities were distínct inter and intra-species. Thrombin-like and pro-coagulant enzymes (activators of Fac- tores II and X) are differently distributed among these venoms. On the other hand, some venoms have only thrombin-like enzyme, such as 8 . atrox (Tucurui) and 8 . atrox (Peru). Others, like 8 . marajoensis, have apparently only pro-coagulant enzymes. 20. ATIVIDADE ANTICOAGULANTE DE TOXINAS DOS VENENOS DE BOTHROPS INSULARIS E BOTH¬ ROPS JARARACUSSU, Heloísa S. Selistre (1); José Roberto Giglio (2); Maria Inês Homsi- -Brandeburgo (3). (1) Departamento de Ciências da Saúde, Universidade Federal de São Carlos, São Carlos, SP. (2) Departamento de Bioquímica, Faculdade de Medicina de Ribeirão Preto — USP, Ribeirão Preto, SP; (3) Departamento de Química e Geociências, IBILCE-UNESP, São José do Rio Preto, SP. Foi estudado 0 efeito de diferentes toxinas sobre o tempo de recalcificação do plasma, utilizando-se plasma citratado de rato. O sangue foi coletado da aorta abdominal de ratos machos de 250g sob anestesia com éter. O tempo de coagulação (TC) foi determinado após incubação do plas- 73 cm 2 3 L. 5 6 11 12 13 14 15 16 Mem, Inst. Butantan, 52 (supl.), 1990 ma com salina ou frações isoladas dos venenos (100 /xg) a 37°C por alguns minutos, cronometrando-se o tempo a partir da adição de 0,1 ml de solução de cloreto de cálcio 0,02M. Das frações testadas do veneno de S. insularis, duas toxinas hemorrágicas (Toxicon 28: 261, 19901 de pesos moleculares próximos e provavelmente diméricas com subunidades diferentes prolongaram sensivelmente o tempo de coagulação, o que não foi observado para a fosfolipase ácida mionecrótica isolada do mesmo veneno. Por outro lado, as fosfolipases ácidas e neutra do veneno de B. jararacussu 40 ui de AB-FUNED, 53.7 ug de L.m.muta consumiram para 100% neut., 8 ul de Al-Butantan e 4 ul de ABL-FUNED. Conclui-se que o critério de incidência de acidentes é insuficiente. Sugere-se que a escolha do ven. de referência se baseie em estudos quantitativos de neutralização cruzada. Apoio financeiro: CNPq-FUNED-UFMG-FAPEMIG 27. COMPARAÇÃO ENTRE TRATAMENTOS DO ACIDENTE BOTRÓPICO COM DIFERENTES DOSES DE ANTIVENENO. Sandra C.B. Castro (1); Ida S. Sano Martins (1); Aura S. Kamiguti (1) Adib S. BouabciU ); Antônio M. Nagamachi!2 ); Domingos M. Cillo!2); Francisco O.S. França!2)-, João L.C. Cardoso!2)\ José C.L. Prado!2 ); Lindioneza A. Ribeiro(2); Miguel T. Jorge! 2); Paulo A. Lei- tão!2): Ronnei A. Ferrari! 2) Rosália P. Leite! 2); Vera B.F. Gualtieri!2 ) (1) Seção de Hematologia e (2) Hospital Vital Brazil, Instituto Butantan, São Paulo, SP. O envenenamento por serpente do gênero Bothrops (acidente botrópico) é responsável pela maioria dos acidentes por serpentes peçonhentas no Brasil. As doses de antiveneno recomendadas no seu tratamento, entretanto, ainda são baseadas principalmente na experiência clínica não con¬ trolada. O objetivo do presente trabalho é avaliar o mais adequado entre dois esquemas terapêu¬ ticos com doses diferentes de antiveneno. Foram selecionados 44 pacientes com envenenamento botrópico leve e moderado e com sangue incoagulável na admissão. Constituíram-se então de forma aleatória dois grupos que receberam antiveneno de ampolas com conteúdo normal ou com apenas a metade da dose (diluído um em dois), em sistema duplo cego: grupo A (n = 26) e grupo B (n = 18). Cada paciente recebeu 4 ou oito ampolas de antiveneno dependendo de o envenenamento ser leve ou moderado, respectivamente. Foram realizados os testes, Tempo de Coagulação, Tempo de Protrombina, Tempo de Trombosplastina Parcial Ativado, Dosagem de Fibrinogênio e Dosagem do Produto da Degradação do Fibrinogênio e Fibrina, na admissão (To) e 6, 12, 24 horas após o término da administração do antiveneno. Os valores médios do fibrinogênio nos tempos To, T6, TI 2 e T24 foram, respectivamente, 0,42; 0,76; 1,03 e 1,42 g/L no grupo A e 0,36; 0,61; 1,05 e 1,45 g/L no grupo B. Esses valores indicam que em ambos os grupos o fibrinogênio atingiu níveis hemostáticos após 12 horas e níveis normais 24 horas após a administração do antiveneno. Os demais testes de coagulação também normalizaram dentro desse período. Os resultados até agora obtidos indicam a eficácia dos dois esquemas terapêuticos aplicados. 28. PROTECTING PROPERTY OF DIDELPHIS ALBIVENTRIS SERUM AGAINST BOTHROPS JARARACA SNAKEVENOM Elen C.T. Landucci! 1); M. Fátima L. Farah (1); José R. Giglio (2); Gilberto B. Domont (3) and Sérgio Marangoni 11) (1) UNICAMP, Depto. Bioquímica, Inst. Biologia, 13081, Campinas, SP. (2) Fac. Medicina Rib. Preto-USP, Depto. Bioquímica, Rib. Preto, 14049, SP (3) UFRJ, Depto. Bioquímica, Inst. Química, Rio de Janeiro, 21910, RJ Previous studies realized by Perales (An.Acad. Cienc., 58, 155, 1986) have shown that Didel- í, | SciELO 76 Mem. Inst. Butantan, 52 (supl.), 1990 phis marsupialis possess a natural immunity against Bothrops jararaca venom. A heterogeneous serie fraction of 50kDa (fTtGE-SDS) from Didelphis serum was described as possessing an anti- hemorragic activity. Three groups of six 20g-mice of swiss strains were injecteo by intraperitoneal route with Bothrops jararaca venom (LD 50 3ug/g) dissolved in PBS solution and mixed with Didel¬ phis serum (ratio 1:1). Controls of Didelphis serum and PBS injections were used to compare with the symptoms observed after 24 hours. Animais injected with Bothrops ve nom - PBS showed characteristic hemorragic symptoms in internai organs as heart, lungs, mesentery, small intes- tine and death after 24 hours. Mice injected with Bothrops venom preincubated with Didelphis serum (30') showed no hemorragic symptoms and all mice survived. These results have shown that the marsupial Didelphis albiventris is resistent to the hemorragic action of Bothrops jararaca venom in the same way as described in other marsupial species. The protection of mice by Didelphis serum indicates that this serie factoris transferible among different species, permiting compari- son of antihemorragic properties in two brazilian marsupial species, D.marsupialis and now D.al¬ biventris. Financial support: CNPq, FAPESP, FIPEC, Instituto Butantan. 29. ATIVIDADE ANTIBOTRÓPICA DE SOROS DE VÁRIOS MARSUPIAIS BRASILEIROS. Perales, J„ Domont, G.B., Ferreira, A.G.C.N., Rocha, S.L.G., Villela, C.G. e Moussatche, H. Departamento de Fisiologia e Farmacodinâmica Fiocruz, Departamento de Bioquímica, I.Q., UFRJ. Rio de Janeiro, Brasil. Departamento de Bioquímica, UCLA, Barquisimeto, Venezuela. Evidenciou-se a resistência de Didelphis marsupialis (80 LD100), Philander opossum (8 LD100), e Lutreolina crassicaudata (3 LD100) ao veneno de Bothrops jararaca (LD100 5/ig/g de camun¬ dongo. i.p.). Mostrou-se também a proteção exercida pelos soros destes marsupiais em camun¬ dongos contra uma LD100 do veneno. Marmosa s.p. não resistiu a uma LD100 de veneno de Bothrops jararaca. Os soros de Marmosa s.p. e Metachirus nudicaudatus não exerceram ação protetora em camundongos. Por cromatografia em DEAE-Sephacel foi isolado do soro de Didel¬ phis marsupialis um complexo proteico antibotrópico (ABF), que após recromatografia (RABF) apresentou, em SDS-PAGE, duas bandas principais de 48,5 e 42,6 KDa, além de duas bandas fracas de 73 e 55 KDa. RABF foi fracionada por cromatografia hidrofóbíca em HPLC (TSK-5PW) e em baixa pressão (Phenyl-Sepharose CL-4B). Nos dois sistemas foram isoladas duas frações: F1P e F2P. Por SDS-PAGE as duas F2P mostraram um perfil similar ao de RABF. A F1P obtida por HPLC evidenciou a presença de duas bandas (48,5 e 73 KDa), e a de baixa pressão uma só banda de 48,5 KDa. F1P não apresentou atividade protetora; F2P mostrou ter atividade anti- botrópica. Estes resultados demonstram que a subunidade de 42,6 KDa é essencial para o exer¬ cício da proteção. Soros de P. opossum e L. crassicaudata cromatografados em DEAE-Sephacel evidenciaram a presença de frações com atividade antibotrópica equivalentes a ABF de D. mar¬ supialis. Por SDS-PAGE essas frações demonstraram possuir perfis semelhantes aos de ABF de Didelphis, com duas bandas principais de 49 e 43 KDa, além de contaminantes menores. A SDS- -PAGE da fração de soro de Metachirus nudicaudatus isolada por DEAE-Sephacel não possui as subunidades de 43 e 49 KDa presentes nos marsupiais resistentes. 30. EFEITOS NA JUNÇÃO NEUROMUSCULAR DO VENENO DE BOTHP™S LANCEOLATUS. Lôbo de Araújo, A.; Donato, J.L.; Leite, G.B.; Rodrigues-Simioni, L. & Praúo-Franceschi, J. — Depto. de Farmacologia — FCM — UNICAMP — Campinas/SP. Através de cromatografia de exclusão e de troca iônica, foram obtidas duas frações com ativida¬ de proteolítica sobre caseína e TAME a partir do veneno bruto de Bothrops lanceolatus. A fração principal, submetida à troca iônica, seguida de um gradiente linear de NaCI originou uma subtra¬ ção com intensa atividade caseinolítica, que se apresentou contaminada por bandas de menor intensidade em eletroforese em gel de poliacrilamida-SDS. Esta subtração, assim como 0 vene¬ no total, mostrou-se muito pouco ativa na preparação nervo-diafragma de camundongo. Entre¬ tanto, quando estudada em biventer cervicis de pintainho (20-40 /ig/ml) determinou contratura seguida de bloqueio das contrações musculares e da resposta induzida pela acetilcolina. Estes efeitos evoluíram para o bloqueio da ordem de 90% ao fim de 120 minutos enquanto que a fra¬ ção fosfolipásica (Lôbo de Araújo, 1990), causou uma redução da amplitude das respostas mus¬ culares de, em média, 20% na dose de 50 /ig/ml durante o mesmo período de tempo. 0 fato da subtração ativa ser capaz de induzir bloqueio das contrações musculares e da contratura pela acetilcolina mas não aquela devida ao potássio, sugere uma ação a nível pós-juncional envol¬ vendo receptores colinérgicos. Lôbo de Araújo, A. — Tese de Doutoramento, 1990 31. MUSCULAR-BLOCKING ACTIVITY OF BOTHROPS MOOJENI VENOM AND ITS ACTIVE FRAC- TIONS. Léa Rodrigues-Simioni ; José Carlos Cogo 1 ; M. T. Assakura 2 and Fajga R. Mandelbaum 2 . Depto. Farmacologia — FCM/UNICAMP — Campinas (SP) 1 . Depto. de Bioquímica — Instituto Butantan — São Paulo (SP) 2 . 77 cm 2 3 z 5 6 11 12 13 14 15 16 Mem. Inst. Butantn, 52 (supl.), 1990 When tested on the chick biventer cervicis preparation, the crude venom of B. moojeni induces muscle contracture followed by irreversible muscular responses blockade at concentrations of 20-80 /ig/ml (n = 5). The components responsible for this effects are located in the main protein peak (Peak II) obtained after fractionation of the crude venom on Sephadex-100 (Assakura eí ai, 1985). Subsequent chromatography of Peak II on DEAE Sephadex A-50 yields 9 fractions. Of these, Fraction 1 shows a similar pharmacological action to the crude venom, viz. muscle contracture followed by irreversible blockade of muscular contraction in response to both elec- tric stimulation and acetylcholine. In contrast, Fraction 2 (80 jig/ml, n = 4) induces a transient (5min) increase in contraction amplitude followed by a decrease in muscular responses. This effect and the Ach contracture are completely reversed by washing the preparation. Fractions 3,4 and 5 also induce moderate muscle contracture and reversible (by washing) blockade. Puri- fied moojeni- Protease A, obtained by further chromatography of Fraction 5, caused muscular responses blockade (80%) but no muscle contracture. However, subsequent to washing of the preparation, intense contracture was observed followed by partial reversal of the muscular blockade. Both the crude venom and all the DEAE Sephadex A-50 fractions obtained from Peak II released creatine kinase from the preparation. We conclude that the contracture caused by the crude venom in the chick biventer cervicis preparation may be caused by the ability of the venom to depolarize muscle fibers as detected in mouse diaphragm isolated preparation. Assakura, M.T.; Reichl, A.P.; Asperti, M.C.A. & Mandelbaum, F.R. Toxicon 23:691, 1985. 32. EFEITOS INDUZIDOS PELO VENENO DE Bothrops insulahs NA PREPARAÇÃO NERVO FRÊNICO- -DIAFRAGMA ISOLADO DE CAMUNDONGO. José C. Cogo; Júlia Prado-Franceschi, Léa Rodrigues-Simioni. Departamento de Farmacologia da Faculdade de Ciências Médicas-UNICAMP. Campinas — São Paulo. Em trabalho anterior, verificamos que o veneno bruto de B. insularisé capaz de induzir bloqueio neuromuscular em preparações isoladas de biventer cervicis de pintainho e nervo frênico dia¬ fragma de camundongos. Este bloqueio, foi geralmente precedido de um leve aumento da am¬ plitude das contrações acompanhado de contratura a qual foi mais acentuada em músculo de aves (Cogo e cols., III Pan-American Symposium on Animal, Plant and Microbial Toxins; p. 45, 1990). No presente trabalho empregando-se preparações isoladas de diafragma de camundon¬ go foi medido o potencial de repouso (RP) da fibra muscular. Verificou-se que concentrações crescentes de 20, 40 e 80 íig/ml determinaram uma gradual diminuição na latência do apareci¬ mento do efeito despolarizante. A concentração mais elevada (80 jig/ml) causa, cinco minutos após sua adição ao banho, uma queda do RP cujo pico máximo estabeleceu-se aos 20 minutos, com valores inferiores a -40 mV (n = 10). A ocorrência dessa queda no RP tanto na região jun- cional como na extrajuncional explica a contratura por nós descrita especialmente na prepara¬ ção de ave. Apoio Financeiro: CAPES, FAP e FAPESP. 33. ALTERAÇÕES MORFOLÓGICAS PRODUZIDAS PELA GUANIDINA NA JUNÇÃO NEUROMUSCU¬ LAR DE CAMUNDONGO. Cruz-Hofling, M.A., Leite, G.B. e Rodrigues-Simioni, L. - "Centro de Microscopia Eletrônica e Departamento de Histologia e Embriologia, I.B., Departamento de Far¬ macologia, FCM/UNICAMP. A guanidina (lOnM) tem um efeito facilitador das contrações musculares que é seguido (em 15(-30') de bloqueio total das respostas do músculo diafragma de camundongo a estímulos elé¬ tricos (Rodrigues-Simioni & Prado-Franceschi, 1989). Após a retirada da droga da preparação por lavagem, observa-se uma facilitação, que em todos os experimentos realizados mostrou um aumento de amplitude das concentrações em geral de 150-350%. Este fato incomum levou- -nos a estudar o fenômeno também através da microscopia eletrônica, com o objetivo de anali¬ sar os elementos estruturais envolvidos nessa complexa ação da guanidina. Diafragmas isolados após a incubação com cloreto de guanidina 10 nM, em solução de Tyrode por 35min., e lavados a seguir, foram preparados para observação ao microscópio eletrônico. A análise ultraestrutural revelou nos fascículos nervosos intramusculares a formação de vacúolos periaxonais interno- dais, que foram interpretados como decorrentes da captação e acúmulo de fluido no espaço entre axoplasma e bainha de mielina. Adicionalmente, as mitocôndrias, tanto as axonais como as do terminal sináptico, como também o próprio terminal mostraram-se edematosos. O núme¬ ro de vesículas sinápticas estava diminuído e mionecrose em diferentes graus foi observada. Al¬ gumas destas alterações assemelham-se àquelas por nós obtidas com venenos que atuam nos canais de sódio. Sugere-se, entretanto, que os efeitos estruturais evocados pela guanidina este¬ jam relacionados ao acúmulo de Ca + + intracelular subsequente ao bloqueio dos canais de K + . Auxílio: FINEP, CNPq, FAPESP, FAP. 34. TsTX-VI AND TsTX-VII. NEW TOXINS FROM Tityus serrulatus SCORPION VENOM. Suely V. Sampaio ^ Eliane C. Arantes ^ Odete A.B. Cunha 2 ; Carlos A. Vieira 2 and José R. Giglio 2 . 78 cm SciELO 10 11 12 13 14 15 16 Mem. Inst. Butantan, 52 (supl.), 1990 11) Faculdade de Ciências Farmacêuticas and (2) Faculdade de Medicina, Universidade de São Paulo, 14049. Ribeirão Preto, SP, Brazil. Fractionation of the "non toxic" pool NT-) (SAMPAIO, et. ai, Toxicon 21,265, 1983) from T. serrulatus venom, on CM-Cellulose-52 afforded six new fractions, CM-I to CM-VI. CM-VI, mol. weight 6,800-7,000 highly toxic and same PAGE mobility and amino acid sequence, up to the 42nd (MARANGONI et ai, unpublished), as 7 -toxin (POSSANI et ai, Biochem. J. 229, 739, 1985) is very probably the same toxin. The complete identity to 7 -toxin, if so, strengths the hypothesis of distribution of a single toxin into two or more chromatographic fractions due to complex for- mation with other proteins. If however minor structural differences are present, we have to as¬ sume that these are responsible for this fractionation, probably due to differences of adsorption on the Sephadex matrix. From CM-I, two new toxins, TsTX-VI and TsTX-VII, were isolated, both of them revealing Gly as N-terminal residue and mol. weights close to 7,000. Their amino terminal sequence are GREGYPADSK and GFIZGYGS, respectively. N-Terminal Gly is uncommon among scorpion toxins and these are the second and third cases found in the venom of T. serrulatus, the first described toxin of this kind being TsTX-ll [TOLEDO and NEVES, Comp. Biochem.Physiol. 558, 249 (1976)]. Most of T. serrulatus toxins have Lys as N-terminal residue. KEY WORDS: Tityus serrulatus — Toxins. Financial support: FAPESP and CNPq. 35. THE EFFECT OF ISOLATED Tityus serrulatus SCORPION VENOM TOXINS ON THE RELEASE OF PUTATIVE NEUROTRANSMITTERS FROM RAT BRAIN CORTICAL SYNAPTOSOMES. Suely V. SampaioJoaquim C. Netto 2 : Eliane C. Arantes '; Odete A.B.Cunha 2 and José R. Giglio 2 . (1) Faculdade de Ciências Farmacêuticas and (2) Faculdade de Medicina, Universidade de São Paulo, 14049 Ribeirão Preto, S.P. Toxins TsTX-VI, TsTX-VII and CM-VI were purified to homogeneity from the "non toxic" frac- tion MT 1 [Sampaio et ai, Toxicon 21,265 (1983)] of T. serrulatus venom by ion-exchange chro- matography on CM-cellulose TsTX-VI (0.3 mg/ 0.3 ml., i.v.) did not evoke in the mouse thetypical symtoms of toxicity of the whole venom, except for a generalized allergic reaction, lacrymation and contracture of the rear legs. The complete amino acid sequence of its 62 amino acid residues (Marangoni et ai, Biochem. J., in press) accounts for a mol. weight of 6,717 and reveals it, together with TsTX-VII (61 aminoacid residues, N-terminal sequence GHZGYGS), as new N-terminal gly toxins. The mobility of TsTX-VII in PAGE is lower than that of TsTX-VI and TsTX-ll, which Is the first reported N-terminal gly toxin from T. serrulatus [Toledo and Neves. Comp. Biochem. Physiol., 55B, 249 (1976)]. By i.v. injection, TsTX-VII evokes the typjcal symptoms of enveno- mation of the whole venom. CM-VI i.v.LD 50 - 0.05 mg/kg, 58 amino acid residues, N-terminal sequence up to the 42nd residue [(Marangoni et. ai, Arq. Biol. Tecnol. 32,146 (1989)] very close to that of 7 -toxin (Possani et. ai, Biochem. J., 229,739, 1985) and the same PAGE mo¬ bility, is apparently identical to 7 -toxin. TsTX-VI, TsTX-VII and CM-VI, similarly to TsTX, (Couti- nho Netto et. ai, J. Neurochem. 35:558, 1980) release glu and 7 -aminobutiric acid (GABA) from rat brain cortical synaptosomes. The "in vitro" release of U 14 - C-Glu and U 14 -C-GABA was studied up to a toxin concentration of 20uM, the maximal release being attained between 10 and 20 uM. The effect of TsTX-VI, CM-VI and the whole venom was ,ocked by tetrodotoxin, thus indicating that it occurs on the sodium channels. Addition of veratrine to CM-VI produced however a synergistic effect. KEY WORDS: Synaptosomes — Toxins — release of neurotransmitters. Financial support: FAPESP and CNPq. 36. AMINO ACID SEQUENCE AND ANTIGENIC STRUCTURE OF TOXIN TsTX-VI FROM THE VENOM OF THE SCORPION TITYUS SERRULATUS. Sérgio Marangoni It); Jorge Ghisoi2): Suely S. Sam¬ paio (3); Eliane C. Arantes (4); José R. Giglio (5); Benedito Oliveira (1) and S/as Frangione (2). (1) UNICAMP, I. Biologia, Dept° Bioquímica, 13.081, Campinas; (2) New York University Medi¬ cai Center, Dept. of Pathology, New York, USA; (3) Dept 0 Anal. Clin. Toxicológicas e Bromatológ- icas; (4) Dept 0 Fis. Química, Fac. Ciências Farmacêuticas de Ribeirão Preto-USP, 14.049 Ribeirão Preto, SP; (5) Dept 0 Bioquímica Fac. de Medicina Rib. Preto-USP, 14.049 Ribeirão Preto S.P. Scorpion toxins constitute a group of low molecular weight proteins that exert pharmacological effects on Na + and K + ion channels. The amino acid sequence of TxTX-VI toxin from the scor¬ pion venom Tityus serrulatus was perfomed from the reduced end S-carboxymethylated toxin, as well as from relevants peptides from the enzymatic digestion which were submitted to Edman automatic sequencial degradation. The primary structure is: GREGYPADSKGCKITCFLTAAGYCN- TECTLKKGSSGYCAWPACYCYGLPESVKIWTSETNKC. This new toxin is the second polypeptide chain from T. serrulatus venom completely sequenced and shows the presence of 62 amino acid residues, a calculated molecular weight of 6717, eight half-cystine residues and no methionine or histidine residues. This results are similar to those of other scorpion toxins. The degree of se¬ quence similarity of TsTX-VI with Ts- 7 (from T. serrulatus venom) is 64%. Hydropathic index JSciELO 79 0 11 12 13 14 Mem. Inst. Butantan, 52'(supl.), 1990 of TsTX-VI was calculated according to Hopp and Woods (Mol. Immunol., 20, 483, 1983). The hydropathic profile of TsTX-VI shows a oontour with same interesting features. Two hydropho- bíc regions isobserved in the polipeptide chain at positions 15-18 and 36-45. The hydrophilioity areas are localized at positions 9-11,32-37 and 57-59, that could keep antigenic properties regions. The primary structure permits homologies studies and help us to localize peptides that contain antigenic determinants that could be used in experimental strategie to obtain molecular vaccines. Neurotoxins and sintetic peptides with Chemical modifications can be used as probe in neurophysio- logic studies (Mol. Immunol., 26, 503, 1989) giving more informations about the relationship among immunological properties, structure and toxicity, of the scorpion toxins. Financial support: CNPq, FAPESP, FIPEC, Instituto Butantan. 37. ESCORPIONISMO NO MUNICÍPIO DE SERTÃOZINHO, SÃO PAULO, Lúcia A. Taveira (1), Cláudio S. Ferreira!!), Maria Esther de Carvalho (1), Vera Regina D. von Eickstedt (3), Francisco Rodri¬ gues (1) & Amaury Lellis Dal Fabbro (1). (1) Superintendência de Controle de Endemias (SUCEN), (2), Dep. Parasitologia, ICB, USP.I3) Seção de Artrópodos Peçonhentos, Instituto Butantan. No município de Sertãozinho, Região Centro-Oeste do Estado de São Paulo, são freqüentes as notificações de encontro de escorpiões (predominantemente Tityus serrulatus ) em prédios resi¬ denciais e outros imóveis. Visando a delinear o perfil da distribuição desses animais na área ur¬ bana, um levantamento inicial foi realizado a partir das notificações recebidas, por um período de dez meses, entre 1988 e 1989. Foram pesquisadas 133 quadras de 32 dos 41 bairros do Município (1,29 quilômetro quadrado ou 1 7,7% da área urbana). As pesquisas foram positivas em 127 quadras (95,5% do total). Ao todo, a pesquisa incluiu 3.011 imóveis (casas, terrenos, etc.), dos quais 397 se mostraram positivos para escorpiões. Destacou-se uma área-controle constituída por sete quadras de onde não se haviam originado notificações durante o período citado. Em quatro dessas quadras foram encontrados escorpiões, o que indica subnotificação. Observaram-se, na maioria dos imóveis examinados, residências ou não, condições favoráveis ao abrigo de escorpiões: entulho, material de construção, mato, ralos danificados, frestas e ou¬ tros. Os moradores entregaram, ao todo, 1.648 escorpiões, dos quais 1.418 mortos. Evidencia- -se a necessidade de um trabalho conjunto com o Instituto Butantan, em São Paulo, para que se possam elaborar estratégias eficientes de controle das populações desses artrópodos. Os ha¬ bitantes do Município deverão ser instruídos sobre a importância da captura de escorpiões vivos e seu encaminhamento para o Instituto Butantan. Deverá ser estimulado o uso das técnicas de captura adequadas. É imprescindível o conhecimento de que os animais vivos são absolutamen¬ te necessários para a obtenção de soro antiescorpiônico. 38. ESTUDO DA ATIVIDADE DERMONECRÓTICA DO VENENO DE LOXOSCELES GAÚCHO, Barbam'. K.C., Ferreira, M.L., Cardoso, J.L., Eickstedt, V.R.D. e Mota, I. Centro de Pesquisa e Formação em Imunologia, Instituto Butantan, São Paulo. Com o objetivo de caracterizar o(s) componente(s) do veneno de L. gaúcho responsável (is) pela atividade dermonecrótica, esse veneno foi submetido a filtração em uma coluna de Sephadex G 100 (70 x 1,8 cm), o que resultou em 3 picos que designamos A, B e C, os quais foram então testados quanto à capacidade de produzir dermonecrose em coelhos. Observamos que a maior parte da atividade dermonecrótica estava localizada no pico A. Esse pico foi então analisado através da técnica de eletroforese em gel de poliacrilamida, na presença de SDS em gel gradiente 5 — 15%. Os resultados mostraram a presença de 2 proteínas majoritárias próximas à região de 30 kDa, que parecem ser as responsáveis pela atividade dermonecrótica. No presente momento estamos realizando experimentos para isolar e caracterizar essas proteínas. * Bolsa de Mestrado FAPESP - Processo 89/1.189-9. 39. ASPECTOS EPIDEMIOLÓGICOS E CLÍNICOS DOS ACIDENTES POR LOXOSCELES SP NO SUL DO BRASIL. Mello da Silva, Carlos A. 11); Torres, João B. 11); Marques, Maria da Graça B. {1); Nicolella, Alberto D.R. 11). (1) Centro de Informação Toxicológica, Sec. Saúde e Meio Ambiente, Porto Alegre, RS. O presente trabalho é resultado de levantamento retrospectivo dos dados constantes nas fichas de atendimento do Centro de Informação Toxicológica (CIT, SSMA/RS), centro regional de in¬ formação e controle de intoxicações para o Estado do Rio Grande do Sul, referentes a acidentes com aranhas do gênero Loxosceles ocorridos durante o ano de 1989. No período foram reportados ao CIT/SSMA, RS 5.424 casos de intoxicação humana por todas as causas dos quais 116 (2,1 %) foram devidos a Loxosceles. Destes, 81 são apresentados e comentados quanto aos aspectos epidemiológicos e clínicos (os 35 casos restantes foram ex¬ cluídos por insuficiência de dados para o estabelecimento de conclusões). A maioria dos casos da amostra ocorreu na área metropolitana de Porto Alegre (72,8%) tendo 19 deles sido avalia¬ dos diretamente pela Equipe (vieram ao CIT). Houve predomínio do sexo feminino (60,4%) e da faixa etária de 20 a 40 anos (46,9%). A região anatômica mais afetada foram os membros í, | SciELO 80 Mem. Inst. Butantan, 52 (supl.), 1990 inferiores (39,5%) e os achados cltnicos predominantes eritema, edema e necrose (acima de 85% dos casos) seguidos de dor local (67,9%). A maioria dos pacientes procurou atendimento nas primeiras 48 horas do acidente (64,1 %) e o tratamento constou de soroterapia antiveneno em 85,1% das vítimas. Outras medidas (debridamento) e reações adversas à soroterapia são analisados. Todos os casos da mostra evoluíram para cura, sendo 9(11,1%) com sequelas locais e 1 (1,2%) desenvolveu insuficiência renal aguda com recuperação posterior (criança 6 anos). Ocorreram 18 casos (22%) considerados graves (sistêmicos) predominando em pacientes acima de 40 anos. Concluímos que o loxoscelismo representa um problema de saúde importante em nossa região, afetando mais ao sexo feminino e com maior gravidade os extremos da vida (crianças e idosos), sendo o loxoscelismo cutâneo a forma de apresentação mais comum no grupo estudado. Aler¬ tamos para o número significativo de reações à soroterapia (-30%) e ao aparente pouco bene¬ fício, quanto à evolução do quadro local, quando usada após 6 a 12 horas do acidente. Estudos prospectivos controlados seriam elucidativos quanto à importância destes aspectos. 40. PRIMEIRO DIAGNÓSTICO DE "LOXOSCELISMO" NA CASUÍSTICA DO HOSPITAL VITAL BRA- ZIL (HVB) J. L. C. Cardoso & D.M. De Cillo Hospital Vital Brazil, I. Butantan. O primeiro diagnóstico clínico do "Loxoscelismo" feito no Brasil teve lugar no ano de 1954 no HVB. Até aquela data as formas necrosantes do araneísmo eram atribuídas à Lycosa. Revendo a seqüência de prontuários do arquivo do HVB, onde o diagnóstico de "Loxoscelismo" foi esta¬ belecido pela primeira vez corresponde ao do paciente F.C. 54 anos, masc.; branco, procedente de Osasco, SP, (PRT. 2351): Admitido ao HVB dia 23/07/1954, com história de que na noite de 21/07, enquanto dormia, foi picado no rosto, não capturando o animal causador do acidente. Ao exame clínico foi constatado edema moderado de hemiface. E, com áreas equimótico-necróticas na pele que recobria a região. Há referência à febre e exantema generalizado, neste dia. No dia 26/07, foi constatada icterícia cutânea e de esclera. Com piora do quadro sistêmico foi transferido para o Hospital das Clínicas da USP em 28/07. Dados de evolução mostram que o paciente foi submetido à cirurgia plástica reparadora, tendo retornado ao HVB para avaliação cerca de 9 meses após a internação. 0 motivo desta apresentação é contribuir para o melhor conhecimento da História do "Loxosce¬ lismo" em nosso País. 41. ESTUDO RETROSPECTIVO DE LATRODECTISMO NA BAHIA fíejâne M. Ura da Silva (1); Gradeia B. Matos (2); fíoney Orismar Sampaio (2). (1) Laboratório de Animais Peçonhentos — UFBA (2) Centro de Informações Antiveneno da Bahia — SESAB Os autores apresentam um estudo retrospectivo dos acidentes com a aranha viúva-negra ILatro- dectus WALCKNAER, 1805) na Bahia na década de 80 a junho de 1990. Nesse período foram registrados no Centro de Informações Antiveneno da Bahia (CIAVE) um total de 19.417 atendi¬ mentos de intoxicação exógena, dentre os quais 64 casos de acidentes por Latrodectus. Apesar da complexidade sistemática do gênero, os autores consideram esse agente etiológico como pertencente ao grupo CURACAVIENSIS. Dentre esses casos, 31 fichas localizadas em arquivo foram revisadas e os sintomas descritos e tabulados, analisando-se sua frequência. Dor local, dor em membros inferiores, tremores e contraturas, pápula eritematosa, dor abdominal e sudorese foram os sintomas mais freqüentes. A idade, o sexo, a procedência dos pacientes e a circunstância dos acidentes são também apre¬ sentados. Estuda-se a evolução dos casos tratados com terapêutica inespecífica — sintomática — e aqueles tratados com soroterapia específica. Descreve-se um caso atendido na emergência do ACRS (CIAVE) com quadro clínico clássico descrito na literatura; o animal foi identificado e o tratamento utilizado foi o soroterápico específico proveniente do Instituto MALBRAM — Argentina. A distribuição geográfica desses animais tem sido verificada principalmente na Região Norte do Estado. No entanto, essa distribuição provavelmente é muito mais ampla, desde quando faltam ainda dados de outros municípios. Na Região Metropolitana de Salvador, a ocorrência da L. cu- racaw'ens/s(MULLER,1776) é frequente na proximidade de residências, favorecendo os acidentes. Os autores sugerem a realização de estudos mais amplos sobre a biologia e distribuição geogrᬠfica desses animais, a clínica e fisiopatologia do acidente, bem como, de caráter imediato, a obtenção do soro específico. Colaboração: Daniel S. Rebouças 42. ACIDENTE POR ARANHAS CHIRACANTHIUM: ASPECTOS EPIDEMIOLÓGICOS E CLÍNICOS. Mi¬ guel T. Jorge (1); Sylvia M. Lucas (2); Vera R. D. Von Eickstedt (2); Lindioneza A. Ribeiro (1) (1) Hospital Vital Brazil, Instituto Butantan (IB) e (2) Seção de Artrópodos Peçonhentos, IB, São Paulo, SP. 81 cm 2 3 z 5 6 11 12 13 14 15 16 Mem. Inst. Butantan, 52 (supl.l, 1990 Foram avaliados 53 casos de acidente por Chiracanthium (Araneomorphae, Clubionidae) atendi¬ dos no Hospital Vital Brazil do Instituto Butantan (HVB-IBI, no peVíodo de 1975 a 1990. Os da¬ dos foram obtidos dos prontuários médicos e o diagnóstico foi realizado pela identificação da aranha na Seção de Artrópodos Peçonhentos do IB. A maioria dos pacientes tinha entre 20 e 49 anos de idade (51%), 30 (57%) pertenciam ao sexo feminino e 23 (43%) ao masculino. Todos os acidentes ocorreram na Grande São Paulo, a maioria no Município de São Paulo (74%). O maior número ocorreu de novembro a abril (83%), quase sempre no período diurno. Dezeno¬ ve pacientes (36%) estavam comendo ou manuseando uvas no momento do acidente e as re¬ giões anatômicas mais frequentemente picadas foram as mãos (21%), os lábios (20%) e a língua (18%). Quarenta e dois (79%) chegaram no HVB dentro de 3 horas após a picada. As manifes¬ tações clínicas mais frequentemente apresentadas foram a dor (98%), o eritema (40%) e o ede¬ ma (38%) no local da picada e as medicações mais comumente utilizadas foram anti-histamínicos (21 %) e analgésicos (9%). A maioria dos pacientes, entretanto, não necessitou de tratamento. Os dados disponíveis evidenciam uma relação entre acidentes por essas aranhas e manipulação de uvas; não se observou em nenhum caso necrose no local da picada, como tem sido relatado em outros países. 43. ACIDENTES POR ARANHAS CARANGUEJEIRAS (MYGALOMORPHAE) ATENDIDOS NO HOSPI¬ TAL VITAL BRAZIL DO INSTITUTO BUTANTAN ENTRE 1966 E 1989. João Luiz Costa Cardoso (1); Sylvia Lucas (2); Pedro Ismael da Silva Júnior (2); Rogério Bertani (3). (1) Hospital Vital Brazil, (2) Divisão de Biologia e (3) Bolsista FUNDAP — Divisão de Biologia, Instituto Butantan, São Paulo, SP. Dados do Hospital Vital Brazil do Instituto Butantan demonstram que as aranhas são responsᬠveis por cerca de 30% dos acidentes ali atendidos (Rosenfeld, G. e Nahas, L. 1976). No período 1966-1989, de um total de 63.390 prontuários, 83 (0,13%) foram de pacientes picados por aranhas caranguejeiras (MYGALOMORPHAE), que trouxeram o animal causador do acidente para identificação. O gênero Acanthoscurria foi o responsável pela maioria dos acidentes (37 casos), seguido de Rachias (14 casos), Pamphobeteus (7 casos), Actinopus (5 casos) e Trechona (1 caso). Em 19 casos não foi possível a identificação a nível de gênero. Do ponto de vista clínico a dor foi o sintoma mais frequentemente observado, isolada ou em associação a outras altera¬ ções (85,54% dos casos); seguido de edema local (26,50%) e eritema (13,25%). Quanto ao tratamento, 45,78% não receberam nenhum tipo de medicação; 21,68% receberam bloqueio local com anestésico; 31,32% foram submetidos a outros procedimentos terapêuticos; um pa¬ ciente recebeu 2 ampolas de soro antiaracnídico. A baixa incidência destes acidentes pode ser explicada pela raridade destes animais nos arredores da capital de São Paulo em relação às ara¬ nhas verdadeiras (ARANEOMORPHAE), como os gêneros Phoneutria, Lycosa, Loxosceles etc., e pelo fato de terem pouco contato com o ser humano devido aos seus hábitos e habitat. 44. ACIDENTES HEMORRÁGICOS POR CONTATO COM TATURANAS NO RIO GRANDE DO SUL (1); Alaour Duarte (2); Irineu Lorini (3); Marco Antonio Schilling (4). (1) Trabalho realizado nos serviços de Nefrologia e Hematologia do Hospital São Vicente de Paulo e no CNPT, EMBRAPA, Passo Fundo, RS; (2) Professor de Nefrologia da Faculdade de Medici¬ na, UPF, Passo Fundo, RS; (3) Entomologista e pesquisador do CNPT, EMBRAPA, Passo Fundo, RS; (4) Serviço de Hematologia do Hospital São Vicente de Paulo, Passo Fundo, RS. No período de janeiro de 1989 a abril de 1990, foram atendidos no Hospital São Vicente de Pau¬ lo, Passo Fundo, 16 pacientes que apresentavam diátese hemorrágica generalizada após conta¬ to com lagartas, posteriormente identificadas como Lonomia (Lepidóptera, Saturniidae). No presente trabalho, são relatadas as manifestações apresentadas por estes pacientes, sua evo¬ lução e resposta ao tratamento usado. Além disso é feita uma descrição da lagarta, sua distribui¬ ção geográfica e população atingida. Algumas considerações são feitas a respeito das ações do veneno baseadas em dados da literatura. 45. SURTO DE DERMATITE POR HYLESIA PAULEX NO LITORAL DO ESTADO DE SÃO PAULO, BER- TIOGA, VERÃO DE 1990. João Luiz C. Cardoso ( 1 ); Tarcísio S. B. Filho (2); Elizabeth C. G. Car¬ neiro (2); Roberto H. P. Moraes (3) (1) Hospital Vital Brazil, Inst. Butantan; (2) Secretaria de Higiene e Saúde de Santos-CCD; e (3) Seção de Parasitologia, I. Butantan. Entre dezembro 1989 e janeiro 1990, foram registrados nos Centros de Saúde e Pronto-Socorro de Bertioga, SP, 612 casos de dermatite pápulo-pruriginosa, de caráter epidêmico, acometendo veranistas e/ou população residente. Coincide o achado com a eclosão maciça de mariposas que, em revoadas noturnas, invadiam os domicílios. Enviadas espécimes do animal ao Prof. Claude Lemaire (Paris), estas foram classificadas como Hylesia paulex Dognin, 1922. Esses pacientes foram tratados, basicamente, com anti-histamínicos por via sistêmica, por pe¬ ríodo variável de 7 a 14 dias, tempo médio de duração das lesões cutâneas. 82 cm SciELO 10 11 12 13 14 15 16 Mem. Inst. Butantan, 52 (supl.), 1990 Concomitantemente, casos semelhantes foram observados em Peruíbe, SP, cidade do litoral, distando cerca de 130 km. de Bertioga. A importância deste relato se deve ao fato de ser esta a terceira descrição desse tipo de patolo¬ gia na literatura brasileira e a primeira no Estado de São Paulo. 46. AÇÃO DO VENENO DE ABELHAS AFRICANIZADAS (VAA) EM RATOS. QUANTIFICAÇÃO DAS CATECOLAMINAS CARDÍACAS. Ferreira, D.B. (1), Oliveira, J.S.M. (1), Costa, /?. S. (1) e Azevedo- -Marques, M. M. (2). (11 Dept? de Patologia e (21 Clínica Médica da Faculdade de Medicina de Ribeirão Preto — USP. Com o objetivo de verificar o comportamento da adrenalina (A) e noradrenalina (NA) do músculo cardíaco de ratos submetidos à ação do VAA, foram inoculados 80 ratos machos Wistar, GE, com 1,5 jil/100g de peso, de VAA, pelas vias SC, IM, IP e EV. No grupo controle (GC), 80 ratos foram inoculados com solução salina 0,15M, pelas mesmas vias e em igual volume. Os GE e GC das IM, IP e SC foram sacrificados por punção cardíaca, sob anestesia, após 4, 7 e 24 horas e os animais da via EV aos 2 minutos. A base do coração foi processada, de acordo com técnica de Anton e Sayre (1968), para extração e quantificação de A e NA. Por leitura espectrofluorimé- trica, a concentração das catecolaminas foi calculada em ^g/g de tecido. Os valores, dados em média amostrai, correspondem às determinações de 8 animais. O teste "t" de Student com p < 0,05 está assinalado, na tabela, com S = diferença estatisticamente significativa e NS = diferença não significativa. Valores de NA e A em g/g de miocárdio. h IM IP SC EV (2 minutos) GE GC t GE GC t GE GC t GE GC t 4 NA 0,480 0,599 NS 0,846 0,882 NS 1,141 0,543 S 0,762 1,034 S A 0,032 0,045 NS 0,068 0,0 S 0,0 0,021 S 0,053 0,0 s 7 NA 0,660 0,610 NS 1.163 0,658 S 1.110 0,998 NS A 0,030 0,028 NS 0,043 0,001 S 0,001 0,0 24 NA 0,611 0,539 NS 0,829 0,744 NS 1,013 0,966 NS A 0,009 0,021 NS 0,005 0,002 NS 0,001 0,0006 NS Conclusões: 1) A via EV mostrou as alterações mais significativas, havendo queda evidente de NA e presença de A no GE. 2) A inconstância dos dados de NA e A obtidos nos animais dos outros GE dificulta a interpretação, parecendo entretanto que o VAA não libera catecolaminas cardíacas neste modelo. Assistência Técnica: A.A. Fujii, A.V. Verceze, U.M. Barros, M. Fteredia, E.P.D. Gual. 47. ESTUDO SOBRE A HIPERINSULINEMIA PRODUZIDA POR CANATOXINA NO RATO. G. Ribeiro- -Da-Silva e J.F. Prado. Departamento de Farmacologia — Faculdade de Ciências Médicas, UNICAMP, Caixa Postal 6111, CEP 13081, Campinas(SP). Canatoxina, uma toxina protéica vegetal (115.000 daltons) extraída das sementes da Canavaiia ensiformis, produz hipoglicemia e hiperinsulinemia no rato. O Sistema Nervoso Autônomo e os opióides endógenos estão envolvidos em vários efeitos produzidos por canatoxina. Como a esti¬ mulação do vago aumenta a insulina plasmática e os opióides endógenos estão envolvidos na secreção deste hormônio, estudamos o efeito do pré-tratamento com hexametônio, atropina, naloxona ou naltrexona, sobre a hipoglicemia e hiperinsulinemia produzidas por canatoxina no rato. Nossos resultados mostraram que houve uma inibição destes dois efeitos da toxina em ra¬ tos pré-tratados com estas drogas. Este fato sugere que há um envolvimento do parassimpático e dos opióides endógenos no aumento de insulina circulante e na hipoglicemia que a canatoxina produz. Auxílios: FINEP (43/87/03/09) e CNPq (401409/86) 48. O ENSINO DOS ACIDENTES POR ANIMAIS PEÇONHENTOS NAS ESCOLAS MÉDICAS BRASI¬ LEIRAS. Benedito Barravieraí') (*) Departamento de Moléstias Infecciosas e Parasitárias da Faculdade de Medicina de Botucatu e Centro de Estudos de Venenos e Animais Peçonhentos da UNESP. O autor avaliou por meio de questionário denominado PROJETO PEÇONHA, o ensino dos aci¬ dentes por animais peçonhentos nas Escolas Médicas brasileiras. Para tanto, em outubro de 1989, enviou 75 questionários para as Faculdades de Medicina, tendo recebido 34 respostas. Em ja¬ neiro de 1990, enviou novamente o mesmo questionário para as demais Escolas e recebeu 17 respostas, totalizando 51 questionários respondidos. Os resultados obtidos permitem considerar que os acidentes por animais peionhentos são atendidos no Hospital Escola em 82,36%. A maioria dos acidentes atendidos é causada por serpentes seguido de aranhas, escorpiões e abelhas. Os alunos do curso de graduação participam em 78,43% deste atendimento, sendo a maioria de- 83 cm 'SciELO 0 11 12 13 14 15 16 Mem. Inst. Butantan, 52 (supl.), 1990 les da 6? série (70,58%), seguidos da 5 a série (52,94%). A maioria dos alunos de graduação (76,47%) tem aulas teóricas, sendo ministradas na 4 a série (76,47%). Estagiam em Institui¬ ções que atendem estes acidentes, 58,22%. Os médicos residentes, 56,86%, recebem treina¬ mento específico, sendo a maioria deles de Clínica Médica (39,21%), seguidos de Doenças Infecciosas e Parasitárias (23,53%). As Disciplinas responsáveis pelo ensino são: Moléstias In¬ fecciosas e Parasitárias (9,80%). Apenas 27,45% destas Disciplinas desenvolvem linhas de pes¬ quisa sobre animais peçonhentos, sendo a maioria na área de serpentes. 49. OCORRÊNCIA DA REAÇÃO SOROLÓGICA NEGATIVA EM PACIENTES COM DIAGNÓSTICO DE ACIDENTE BOTRÓPICO. Domingos,M.C; Cardoso, J.L.C; Mota, I. Hospital Vital Brazil e Centro de Pesquisa e Formação em Imunologia, Instituto Butantan — São Paulo — SP. Sessenta pacientes com diagnóstico de acidente botrópico atendidos no Hospital Vital Braz' 1 submetidos a soroterapia, foram estudados para a pesquisa de anticorpos humorais antiveneno. Este estudo foi realizado através do teste Elisa em amostras coletadas no intervalo de 18 dias a 1 ano após a picada. Os dados obtidos revelaram que 56% dos pacientes apresentaram títulos positivos. Como a técnica proposta para inquérito epidemiológico tem sido a detecção de anticorpos pre¬ sentes no soro, os dados aqui apresentados indicaram a necessidade de um estudo mais apro¬ fundado, sobre a soroconversão em pacientes picados, bem como do estabelecimento de grupos controles. 84 cm SciELO 10 11 12 13 14 15 16 AUTHORS INDEX I — PLENARY SESSIONS Name, page n? AMARAL, C.F.S., 27 AZEVEDO-MARQUES, M.M 27 BUCARETCHI, F., 33, 61 CARDOSO, J.L.C., 43 CUPO, P., 27 DINIZ, C.R., 49 FONTANA, M.D., 59 FRANÇA, F.O.S., 63 FREIRÉ-MAIA, L., 51 GUTIERREZ, J.M., 37 HAWGOOD, B.E., 21 HERING, S., 27 KOUYOUMDJIAN, J.A. 45 LAURE, C.J., 17 MANDELBAUM, F., 35 PINTO, R.N.L., 47 PRADO-FRANCESCHI, J, 25 41 RIBEIRO, L.A., 15 SANO-MARTINS, I.S., 39 VITAL-BRAZIL, O., 23, 31 II - POSTERS SESSION Name, Abstract n? ANDRADE, S.R.M. 10 ANDRADE JR., H.F. 5 ARANTES, E.C. 34, 35, 36 ASSAKURA, M.T. 9, 19, 31 AZEVEDO-MARQUES, M.M. 14, 46 BARBARO, K.C. 38 BARRAVIERA, B. 48 BELTRAN, J.R. 4 BERTANI, R. 43 BOUABCI, A.S. 27 CARDOSO, D.F. 12 CARDOSO, J.L.C. 3, 27, 38, 40 43 45,49 CARNEIRO, E.C.G. 45 CARVALHO, M.E. 37 CASTRO, S.C.B. 3,21,27 CHUDZINSKI, A.M. 21 CILLO, D.M. 27,40 COGO, J.C. 7,31,32 COSTA, R.S. 14,46 CRUZ, M.C.G. 5 CRUZ-HOFLING, M.A. 33 CUNHA, O.A.B. 34,35 CURY, Y. 13 DINIZ, C.R. 10,18,25,26 DINIZ, M.R.V. 6 DOMINGOS, M.C. 49 DOMINGUES DO CARMO, L.C.T.25,26 DOMONT, G.B. 28,29 DONATO, J.L. 30 DUARTE, A. 44 EICKSTEDT, V.R.D. 37,38,42 FABBRO, A.L.D. 37 FAN, H.W. 3 FARAH, M.F.L. 28 FERRARI, R.A. 27 FERREIRA, A.G.C.N. 29 FERREIRA, C.S. 37 FERREIRA, D.B. 14, 46 FERREIRA, M.L. 38 FERREIRA-ALVES, D.L. 10,18 25 26 FILHO, T.S.B.45 FORTES DIAS, C.L. 13 FRANÇA, F.O.S. 3, 27 FRANGIONE, B. 36 FREITAS, J.C. 1 FURTADO, M.F.D. 8,13,19 GHISO, J. 36 GIGLIO, J.R. 15,20,28,34,35,36 GONÇALVES, L.R.C. 13 GUALTIERI, V.B.F.27 GUTIERREZ, J.M. 7,13 HAWGOOD, B.E. 2 HOMSI-BRANDEBURGO, M.L 15, 20 JORGE, M.T. 27,42 KAMIGUTI, A.S. 3,21,27 KELEN, E.M.A. 21 KOUYOUMDJIAN, J.A. 22 LANDUCCI, E.C.T. 28 LAURE, C.J. 4 LEITÃO, P.A. 27 LEITE, G.B. 30, 33 LEITE, R.P. 27 LOBO DE ARAÚJO, A. 30 LORINI, L, 44 LUCAS, S.M. 42, 43 MAIA, P.Z. 23 MANDELBAUM, F.R. 9,16,19,31 MARANGONI, S. 28, 36 MARQUES, M.G.B. 39 MARTINS, M.M. 10 MATOS, G.B. 41 MATOS, M.F.C. 16 MELLO DA SILVA, C.A. 39 MELO, P.A. 15 85 cm 'SciELO 0 11 12 13 14 15 16 MORAES, R.H.P. 45 SAMPAIO, M.U. 16 1 MORENO, R.A. 11 SAMPAIO, R.O. 41 1 MOTA, 1. 38, 49 SAMPAIO, S.S. 36 1 MOURA DA SILVA, A.M. 12 SAMPAIO, S.V. 34, 35 I MOUSSATCHE, H. 29 SANO-MARTINS, I.S. 21, 27 1 I NAGAMACHI, A.M. 27 SANTORO, M.L. 21 1 NASCIMENTO, N. 5 SANTOS, M.C. 13 1 NETTO, J.C. 35 SANTOS, S.M. 6 1 NICOLELLA, A.D.R. 39 SAWAYA, P. 8 1 I OLIVEiRA, B. 36 SCHILLING, M.A. 44 1 OLIVEIRA, J.R.F. 3 SELISTRE, H.S. 20 I OLIVEIRA, J.S.M. 46 SERRANO, S.M.T. 16 j PERALES, J. 29 SILVA, D.G. 22 1 POLIZELLI, C. 22 SILVA, F.R.S.V. 23 I PRADO, J.C.L. 27 SILVA, R.M.L. 41 I PRADO, J.F. 47 SILVA JR., P.l. 43 PRADO-FRANCESCHI, J. 7,11, SILVEIRA, U.S. 6 1 17,30,32 SUAREZ-KURTZ, G. 15 PUORTO, G. 8, 9 TANIZAKI, M.M. 12 RANIERI, D. 22 TAVEIRA, L.A. 37 1 RIBEIRO, L.A. 27, 42 TERUYA, K. 23 RIBEIRO DA SILVA, G. 47 TOMAC JR., U. 5 ROCHA, S.L.G. 29 TOMY, S.C. 21 RODRIGUES, F. 37 TORRES, E.M. 24 RODRIGUES-SIMIONI, L. 7, 30, TORRES, J.B. 39 31,32,33 VIEIRA, C.A. 34 ROGERO, J.R. 5 VILLELA, C.G. 29 SADALA, L. 18 ZAMBELLI, J.E. 11 SALOMÃO, M.G. 8, 9 ZANIN, R.C.S. 23 SAMPAIO, C.A.M. 16 ZAPPELLINI, A. 17 1 86 cm SciELO 10 11 12 13 14 15 16 COMPOSIÇÃO. FOTOLITO E IMPRESSÃO IMPRENSA OFICIAL DO ESTADO SAIMESP Rua da Mooca, 1921 — Fone: 291-3344 Vendas, ramais: 257 e 325 Telex: 011-34557 — DOSP Caixa Postal: 8231 — São Paulo C.G.C. (M.F.) N.° 48.066.047/0001-84 N0V0 TEMPO TRABALHO E DESENVOLVIMENTO JSciELO, 6 INSTRUÇÕES AOS AUTORES 1. Somente serão aceitos trabalhos inéditos e que se destinem exclusivamente à revista. É proibida a reprodução com fins lucrativos. Os artigos de revisão serão publicados a convite da Comissão Editorial. 2. Os trabalhos deverão ser redigidos em português, inglês ou francês, datilografados prcferencialmente em mᬠquina elétrica, em espaço duplo em 3 (três) vias, em papel formato ofício e numerados no ângulo superior direi¬ to. 3. No preparo do original será observada, sempre que possível, a seguinte estrutura: Página de rosto: título do arti¬ go, nome(s) do(s) autor(es) e filiação científica. Texto: introdução, material c métodos, resultados, discussão, conclusões, agradecimentos c referência bibliográfica. Material de referência: resumos (em português e inglês); unitermos (palavras ou expressões que identificam o conteúdo do artigo; devem ser incluídas até um limite máxi¬ mo de três. cm português e inglês). 4. As referências bibliográficas deverão ser ordenadas alfabeticamente e numeradas. Excmpltjs: Para livros: autor, título, edição, local de publicação, editor, ano. páginas. 7. BIER, O. Microbiologia e imunologia. 24.ed. São Paulo, Melhoramentos, 1985 1234p. Para artigos: autor, título do artigo, título do periódico, volume, página inicial e final, ano. 8. MACHADO, J.C. & SILVEIRA F.°, J.F. Obtenção experimental da pancreatite hemorrágica aguda no cão por veneno escorpiônico. Mem. Inst. Butantan, 40/41: 1-9, 1976/77. As citações no texto devem ser por números-índices correspondentes às respectivas referências bibliográficas. Exemplos: ... método derivado de simplificação de armadilha de Disney 1 ... segundo vários autores 2 3 ' 4 5. As ilustrações (fotos, tabelas, gráficos etc.) deverão ser originais c acompanhadas de legendas explicativas. As le¬ gendas serão numeradas e reunidas em folha ã parte. Os desenhos deverão ser a nanquim e as fotografias bem nítidas, trazendo no verso o nome do autor c a indicação numérica da ordem a ser obedecida no texto. As ilustra¬ ções deverão ser organizadas de modo a permitir sua reprodução dentro da mancha da revista (22 x 12,5cm). 6. Os artigos deverão conter no máximo 6 (seis) ilustrações (branco e preto). De cada trabalho serão impressas 50 (cinquenta) separatas, sendo 10 para a Biblioteca do Instituto e 40 para os autores. 7. Os textos originais não serão devolvidos e os originais das ilustrações estarão à disposição dos autores. INSTRUCTIONS TO AUTHORS 3. 4. 1. Manuscripts submitted to the Editorial Board should bc unpublished texts and should not bc under considera- tion for publication clsewhere. Reproduction for commercial purposes is not allowed. The Editorial Board wi 11 plan the publication of revision articles. 2. The original and two copies of papers should bc typewritten in Portuguese, English or French, double spaced, on typing paper (31 x 21cm). Pages should be numbered consecutivcly at the upper rigjtt corner. The following structure should be considered in the preparation of the manuscript: Title page: with article ti- tlc, name of author(s), profcssional address. Text: with introduction, material and mcthods. rcsults. discus- sion, conclusions, acknowlcdgments, references, abstraets (in Portuguese and English), and keywords. A maximal number of 03 keywords should bc included in Portuguese and English. References in alphabctical order should be numbered consecutivcly. Examplcs: Books 7. BIER, O. Microbiologia c imunologia. 24.ed. São Paulo, Melhoramentos, 1985. 1234p. Articles 8. MACHADO, J.C. & SILVEIRA F.°, J.F. Obtenção experimental da pancreatite hemorrágica aguda no cão por veneno escorpiônico. Mem. Inst. Butantan, 40/41: 1-9, 1976/77. Citations in the text should bc identified by the refercnce number. Examplcs: ... método derivado de simplificação de armadilha de Disney 1 ... segundo vários autores 2 5. Illustrations (photographs, tablcs, figures etc.) should be the originais and legends should bc submitted typcw- ritten on a separate shcct. Linc-drawings should bc with China ink and photographs must be of top quality. On the back of cach figure or photograph the name of the author(s) should be lighly written and the number indi- cating the scqucnce in the text. Illustrations should fit in a page measuring 22 x 12,5cm. 6. No more than 6 illustrations will bc acccptcd and photographs should bc black and white. Fifty reprínts of each article are provided without charge, and 10 will be kept at the library. 2. Submitted manuscripts will not be rcturned to the author(s) but the original illustrations are available to au- thor(s) by request. cm iSciELO 0 11 12 13 14 15 16