The interaction of bothropstoxin-I (Lys49-PLA(2)) with liposome membranes

Bothropstoxin-I (BthTx-I) is a Lys49-PLA(2) from the venom of the snake Bothrops jararacussu, which permeabilizes biological and artificial membranes by a mechanism independent of lipid hydrolysis. This mechanism has been investigated by studying the interaction of nine single tryptophan BthTx-I mutants with negatively charged phospholipid membranes. Changes in the solvent exposure of the tryptophan in each mutant were evaluated comparing the rate of chemical modification (k(mod)) by bromosuccinamide with the maximum intrinsic tryptophan fluorescence emission wavelength (lambda(max)) in buffer and in the presence of 10% DMPA/90% DPPC liposomes. No changes in lambda(max). were observed, whereas k(mod) values for tryptophans at positions 7, 10, 31 and 125 were significantly reduced in the presence of lipids, suggesting that bound phospholipid decreases solvent accessibility at these positions. Since the half-lives of the fluorescence and chemical modification effects differ by at least six orders of magnitude, these results suggest that the bound phospholipid may interact with multiple locations on the protein surface over micro- to millisecond timescales. (C) 2009 Elsevier Ltd. All rights reserved.

Reduced pH induces an inactive non-native conformation of the monomeric bothropstoxin-I (Lys49-PLA(2))

Bothropstoxin-I (BthTx-I), a Lys49-PLA(2) from Bothrops jararacussu venom, permeabilizes membranes by a non-hydrolytic Ca(2+)-independent mechanism. The BthTx-I showed activity against liposomes including 10% and 50% negatively charged lipids at pH 7.0, but not at pH 5.0. Nevertheless, ultracentrifugation and FRET demonstrated that at pH 5.0 the BthTx-I is bound to 50% negatively charged membranes. ANS binding identified a non-native monomeric conformation at pH 5.0, suggesting that tertiary structure alterations result in activity loss of the BthTx-I at low pH. (C) 2009 Elsevier Ltd. All rights reserved.

Bites by the colubrid snake Philodryas patagoniensis: A clinical and epidemiological study of 297 cases

We retrospectively analyzed 297 proven cases of Philodryas patagoniensis bites admitted to Hospital Vital Brazil (HVB), Butantan Institute, Sao Paulo, Brazil, between 1959 and 2008. Only cases in which the causative animal was brought and identified were included. Part of the snakes brought by the patients was still preserved in the collection maintained by the Laboratory of Herpetology. Of the 297 cases, in 199 it was possible to describe the gender of the snake, and seventy three (61.3%) of them were female. The length of snakes (snout-vent length) ranged from 160 to 1080 mm. In 117 snakes their state of preservation enabled the dissection and examination of their stomach contents. The stomach was empty in 106 snakes (89.1%). Most bites occurred in the seasons of spring and summer (n = 196, 66.0%) and during warmer periods of the day. The mean age of the victims was 24.1 +/- 15.1 years old and 206 (69.4%) patients were men. Around 92% of the patients sought medical care within 6 h after the bite. Both lower (n = 188, 63.3%) and upper limbs (n = 102, 34.3%) were most frequently bitten, especially the feet and hands (n = 205, 69.0%). The local clinical manifestations were pain (n = 151, 50.8%), transitory bleeding (n = 106, 35.7%), erythema (n = 47, 15.8%) and edema (n = 39, 13.1%). Ecchymosis was not observed. Only 7 (2.4%) patients reported systemic symptoms characterized by mild dizziness and 88 patients (29.6%) showed no evidence of envenoming. The whole blood clotting time was performed in 76 (25.6%) patients on admission and all of them had coagulable blood. Supportive treatment was offered to only 13.4% of patients, namely administration of antihistamines (n = 19, 6.4%) and analgesics (n = 12, 4.1%). Eight patients (2.7%) were mistreated with Bothrops antivenom before their admission to HVB. No sequels or relevant complications were observed in patients, and the prognostic was benign. Therefore, although P. patagoniensis accidents can cause mild local symptomatology, it is very important that health professionals know how to make the correct diagnosis to avoid unnecessary use of antivenom. (C) 2010 Elsevier Ltd. All rights reserved.

Inhibition of Snake Venoms and Phospholipases A(2) by Extracts from Native and Genetically Modified Eclipta alba: Isolation of Active Coumestans

We genetically modified Eclipta alba using Agrobacterium rhizogenes LBA 9402, with the aim of producing secondary metabolites with pharmacological properties against phospholipase A(2) and the myotoxic activities of snake venom. Extracts from in natura aerial parts and roots, both native and genetically modified (in vitro), were prepared and analysed by high-performance liquid chromatography. In natura materials showed the coumestan wedelolactone at higher concentration in the aerial parts, while demethylwedelolactone appeared at higher concentration in roots. Among the modified roots, clone 19 showed higher concentrations of these coumestans. Our results show that the in natura extracts of plants collected from Botucatu and Ribeirao Preto were efficient in inhibiting snake venom phospholipase A(2) activity. Regarding in vitro material, the best effect against Crotalus durissus terrificus venom was that of clone 19. Clone 19 and isolated coumestans (wedelolactone and demethylwedelolactone) inhibited the myotoxic activity induced by basic phospholipases A(2) isolated from the venoms of Crotalus durissus terrificus (CB) and Bothrops jararacussu (BthTX-I and II). The search for antivenom is justified by the need of finding active principles that are more efficient in neutralizing snake venoms and also as an attempt to complement serum therapy.

Venom-Sweet-Venom: N-Linked Glycosylation in Snake Venom Toxins

Protein glycosylation represents one of the most important post-translational events, and is a mean of diversifying a protein without recourse to the genome. The venoms produced by snakes contain an abundance of glycoproteins with N-linked carbohydrates. N-linked glycosylation can ensure the correct folding of important functional domains. Characterization of carbohydrates structures aids in development of human therapeutics by snake venom toxins.

Antibothropic action of Casearia sylvestris Sw. (flacourtiaceae) extracts

Casearia sylvestris Sw., popularly known in Brazil as `guacatonga`, has been used as antitumor, antiseptic, antiulcer, local anaesthetic and healer in folk medicine. Snakebite envenomation by Bothrops jararacussu (Bjssu) constitutes a relevant public health hazard capable of inducing serious local damage in victims. This study examined the pharmacological action of apolar and polar C sylvestris leaf extracts in reverting the neuromuscular blockade and myonecrosis, which is induced by Bjssu venom and its major toxin bothropstoxin-I on the mouse phrenic nerve-diaphragm preparations. The polar methanol extract (ME) was by far the most efficacious. ME not only prevented myonecrosis and abolished the blockade, but also increased ACh release. Such facilitation in neuromuscular transmission was observed with ME alone, but was accentuated in preparations incubated with ME plus venom or toxin. This established synergy opens an interesting point of investigation because the venom or toxin in contact with ME changes from a blocking to a facilitating effect. It is suggested that rutin, known to have potent antioxidant properties, and one of the components present in the ME, could have a role in the observed effects. Since commercial rutin did not reproduce the ME effects, it is likely that a rutin-containing phytocomplex is neutralizing the bothropic envenoming effects. Copyright (C) 2008 John Wiley & Sons, Ltd.

Epidemiologia do acidente por serpentes peçonhentas: estudo de casos atendidos em 1988

Foram avaliados aspectos epidemiológicos de acidentes por serpentes peçonhentas no Estado de São Paulo, Brasil, com base em prontuários de 322 pacientes e em entrevistas feitas com 209 deles e/ou seus acompanhantes. Os acidentes ocorreram principalmente com pessoas de 10 a 20 anos de idade, do sexo masculino, nos meses de outubro a abril e no período diurno. As regiões anatômicas mais freqüentemente picadas foram os pés, as mãos e as pernas. Bothrops, Crotalus e Micrurus foram responsáveis por, respectivamente, 95,0%, 4,4% e 0,6% dos casos. Não ocorreram óbitos, mas 2,2% dos pacientes apresentaram seqüelas. Dentre os 209 entrevistados, a ocupação de lavrador foi a mais freqüentemente relacionada ao acidente que, em aproximadamente 60% das vezes, ocorreu durante o trabalho. O total de 160 pacientes (76,6%) submeteram-se a alguma forma de tratamento antes de chegarem a um serviço de saúde: foram mais comuns o uso de torniquete (50,2%), a expressão local na tentativa de retirar parte do veneno (33,5%), a colocação das mais diversas substâncias sobre o local da picada (36,8%) e a ingestão de outras (12,9%); pouco mais de um quarto dos pacientes submeteram-se a alguma forma de tratamento médico antes de chegar ao HVB sendo mais comum a antissepsia (8,2%), a administração do antiveneno (6,2%), de anti-histamínicos (5,7%) e de analgésicos (5,3%).

Vulnerabilities in snakebites in Sao Paulo, Brazil

ABSTRACT OBJECTIVE To describe elements of vulnerability of victims of snakebite. METHODS This qualitative, descriptive, cross-sectional study had, as theoretical framework, the concept of vulnerability in individual, social, and programmatic dimensions. We interviewed 21 patients admitted into a hospital specialized in the care of accidents caused by venomous animals. The interviews were analyzed according to a discourse analysis technique. RESULTS Patients were mainly young men, living in remote countryside areas, where health services frequently have limited resources. We found social and individual conditions of vulnerability, such as precarious schooling, low professional qualification, housing without access to piped water, no sewage treated, and no regular garbage collection, and lack of knowledge on this health problem. Regarding the programmatic dimension, we found limited accessibility to the health services that could affect the prognosis and the frequency of sequelae and deaths. CONCLUSIONS Considering such vulnerabilities evoke the need to improve the program for control the Accidents by Venomous Animals and the training of health workers, we highlight the potential use of the concept of vulnerability, which may amplify the understanding and the recommendations for the practice and education related to snakebites.

Insuficiência renal aguda secundária a acidentes ofídicos botrópico e crotálico. Análise de 63 casos

Sessenta e três pacientes com insuficiência renal aguda secundária a acidente ofídico foram tratados no CTI do Hospital das Clínicas da UFMG. Em 32 pacientes (51%) o acidente foi produzido por serpentes do gênero Bothrops (grupo bio-trópico) e em 32 pacientes (49%) pela cascavel sul-americana (grupo crotálico). As principais complicações apresentadas pelos pacientes foram a uremia (100% dos casos), hiperpotassemia (89% dos casos), anemia (78% dos casos), infecção urinária (37% dos casos), hiper-hidratação (17% dos casos), parada cardíaca (14% dos casos) e edema agudo dos pulmões (11% dos casos). Cinco pacientes do grupo crotálico (16%) tiveram insuficiência respiratória aguda atribuída à ação neurotóxica do veneno, quatro dos quais se recuperaram completamente. Sete pacientes do grupo botrópico (22%) tiveram necrose cortical renal diagnosticada em cinco através da biópsia renal e em dois na necropsia. Quarenta e cinco pacientes (71%) foram tratados com diálise peritoneal e a hemodiálise foi necessária em dois pacientes, um dos quais havia sido submetido a diálise peritoneal. Em 17 pacientes (27%) o tratamento foi conservador. Cinqüenta e cinco pacientes receberam alta hospitalar, quatro dos quais com insuficiência renal crônica secundária a necrose cortical renal e oito (13%) faleceram. Os óbitos foram atribuídos a edema pulmonar agudo em quatro pacientes, a estado de choque em dois pacientes e a coma e infecção respiratória após parada cardíaca em dois pacientes.

Methemoglobinemia associated with loxoscelism

In twenty five patients who presented the cutaneous form of loxoscelism, serum haptoglobin and lactic dehydrogenase, erythrocyte glucose-6-phosphate dehydrogenase, glutathione reductase, glutathione peroxidase, methemoglobin, bilirubin and reticulocytes were investigated after bite. No hemolysis was detected but an increase in methemoglobin was found in 54% of the cases; in 7% it was between 1.1% and 2%, in 27% it ranged from 2.1% to 4%, and in 20% from 4.1% to 8%. Blood samples of a normal, blood group 0 individual and of a patient who exhibited methemoglobinemia after Loxosceles bite were incubated separately with antisera against Loxosceles gaucho, Crotalus terrificus, Bothrops jararaca, with Loxosceles gaucho venom and 0.3% phenol. No methemoglobin was found after 1, 4,8 and 15 days in both sets of samples. At the 25th day all the samples, including the controls, exhibited similar methemoglobin reductase decrease. The data suggest that the methemoglobinemia which occurs in 50% of the patients probably arises from in vivo venom metabolism, inasmuch as the crude venom does not induce methemoglobinemia.

Non-venomous snake bite and snake bite without envenoming in a brazilian teaching hospital: analysis of 91 cases

A retrospective survey of 473 cases of snake bite admitted to a Brazilian teaching hospital from 1984 to 1990 revealed 91 cases of bite without envenoming and/or caused by non-venomous snakes. In 17 of these cases the snake was identified, and one patient was bitten by a snake-like reptile (Amphisbaena mertensii). In 43 cases diagnosis was made on clinical grounds (fang marks in the absence of signs of envenoming). The other 30 cases were of patients who complained of being bitten but who did not show any sign of envenoming or fang mark. Most cases occurred in men (66;73%), in the 10-19 years age group (26;29%), in the lower limbs (51/74;69%), between 6 A. M. and 2 P.M. (49;61%) and in the month of April (16; 18%). One patient bitten by Philodryas olfersii developed severe local pain, swelling and redness at the site of the bite, with normal clotting time. The patient bitten by Drymarcon corais was misdiagnosed as being bitten by a snake of the genus Bothrops, was given the specific antivenom, and developed anaphylaxis. One patient bitten by Sibynomorphus mikanii presented prolonged clotting time, and was also given antivenom as a case of Bothrops bite. Correct identification of venomous snakes by physicians is necessary to provide correct treatment to victims of snake bite, avoiding unnecessary distress to the patient, and overprescription of antivenom, which may eventually cause severe untoward effects.

Ineficácia do antiveneno botrópico na neutralização da atividade coagulante do veneno de Lachesis muta muta: relato de caso e comprovação experimental

No Estado do Amazonas, nas regiões circunvizinhas à cidade de Manaus, as principais espécies de serpentes causadoras de acidentes são Bothrops atrox e Lachesis muta muta com um percentual de ocorrência, dos acidentes confirmados, de 76% e 17%, respectivamente. Rotineiramente, na ausência dos soros antilaquético e antibotrópico-laquético o Instituto de Medicina Tropical de Manaus (IMTM), utiliza-se do soro antibotrópico no tratamento do acidente laquético. Neste trabalho relatamos um caso de acidente por L m. muta, onde o paciente foi tratado com 20 ampolas do soro antibotrópico e permaneceu com o sangue incoagulável até o 13º dia após o acidente. Experimentos foram realizados para obtenção das potências do soro antibotrópico para as atividades coagulante e hemorrágica dos venenos de L. m. muta e de B. atrox. Os resultados mostram que as potências do soro para a atividade hemorrágica dos venenos de L. m. muta e de B. atrox foram similares enquanto que a potência, para a atividade coagulante do veneno de L. m. muta, foi 9,2 vezes menor. Os títulos de anticorpos de três diferentes lotes de soro antibotrópico variaram para o veneno de L. m. muta e foram constantes para o veneno de B. atrox. Devido a ineficácia do soro antibotrópico em neutralizar, principalmente, a atividade coagulante do veneno de L. m. muta, sugerimos a não utilização do antibotrópico no tratamento dos acidentes por L. m. muta.

Philodryas patagoniensis bite and local envenoming

A 5-year-old boy bitten by a specimen of Philodryas patagoniensis, a colubrid snake currently classified as nonvenomous, developed signs of local envenoming characterized by swelling and warmth on the bitten limb. This is the first time that local envenoming following Philodryas patagoniensis bite is recognized. Based on the clinical findings and misidentification of the snake, the patient was treated as a victim of Bothrops bite, having received unnecessarily the specific antivenom. Educational efforts to make doctors and health workers capable to identify correctly venomous snakes are necessary, to avoid inappropriate indication of antivenom and decrease the risk of its potentially harmful untoward effects. Examination of the bite site can be useful to the differential diagnosis between pit viper and colubrid bites.

Liver dysfunction in patients bitten by Crotalus durissus terrificus (Laurenti, 1768) snakes in Botucatu (State of São Paulo, Brazil)

Thirty-two patients bitten by venomous snakes sixteen by Bothrops spp. and sixteen by Crotalus durissus terrificus were studied. The group comprised thirty males and two females, aged eight to sixty-three years (mean 33±15). Bromsulphalein tests were increased in the majority of patients bitten by Crotalus durissus terrificus. The correlation coefficient of Spearman was positive between bromsulphalein tests and alanine aminotransferase levels, and between alanine aminotransferase and aspartate aminotransferase levels only in the Crotalus group. The only patient who died was bitten by Crotalus durissus terrificus and showed hydropic degeneration and mitochondrial injury in the liver. It was concluded that the hepatic damage might have been caused by at least two possible mechanisms: venom effect on liver mitochondria and cytokine effects on hepatocyte, specially interleukin-6.

Epidemiological and clinical aspects of snAkebite in Belo Horizonte, Southeast Brazil

Epidemiologic and clinical aspects of 310 hospitalized snakebite patients and 310 matched controls were described, over a seven years period, from an emergency hospital in Belo Horizonte, Southeast Brazil. The diagnosis was based upon clinical picture or actual snake identification. Fifty six percent of victims were bitten by the snakes of genus Bothrops, 32.0% by Crotalus, 1.0% by Lachesis and 10.0% undetermined. During the study period, stable number of cases and marked seasonal variation were noted. In comparing cases of snakebite and controls, those from a rural area or who were involved in agricultural labor activity were identified as a high risk group, with an odds ratio (OR) of 14.7 and 6.7, respectively, in favor of being bitten. Upon treatment, snakebite patients were 13.5 times more likely to have had early anaphylactic reactions than their controls, with a higher association in the age group ³ 20 years (OR = 30.3). Increased risks were also detected for pyrexia (OR = 11.7), with a marked association in the group under 19 years old (OR = 16.6). Severe cases of snakebite are an important treatable cause of morbidity in Brazil but therapy may be potentially life threatening. The higher case-fatality ratio encountered, compared to national statistics may be due the representativeness of the more severe cases who sought hospitalization. Preventing snakebite and early referral of those who are bitten is proposed