RESUMO
Snake envenomation is a neglected tropical disease. In Brazil, the Bothrops genus is responsible for about 86% of snakebite accidents. Despite extensive evidence of the cytotoxicity of snake venoms, the cellular and molecular mechanisms involved are not fully understood, especially regarding the effects on cell cycle progression and cytoskeleton organization. Traditionally, the effectiveness and quality control tests of venoms and antivenoms are assessed by in vivo assays. Despite this, there is a rising effort to develop surrogate in vitro models according to the 3R principle (Replacement, Reduction, and Refinement). In this study, we treated rat liver cells (BRL-3A) with venoms from five Bothrops species (B. jararaca, B. jararacussu, B. moojeni, B. alternatus, and B. neuwiedi) and analyzed cell viability and IC50 by MTT assay, cell cycle phases distribution by flow cytometry, and morphology and cytoskeleton alterations by immunofluorescence. In addition, we evaluated the correlation between IC50 and the enzymatic and biological activities of each venom. Our results indicated that Bothrops spp. venoms decreased the cell viability of rat liver BRL-3A cells. The rank order of potency was B. jararacussu > B. moojeni > B. alternatus > B. jararaca > B. neuwiedi. The mechanisms of cytotoxicity were related to microtubules and actin network disruption, but not to cell cycle arrest. No clear correlation was found between the IC50 and retrieved literature data of in vitro enzymatic and in vivo biological activities. This work contributed to understanding cellular and molecular mechanisms underlying the Bothrops spp. venom cytotoxicity, which can help to improve envenomation treatment, as well as disclose potential therapeutic properties of snake venoms.
RESUMO
Snake envenomation is a neglected tropical disease. In Brazil, the Bothrops genus is responsible for about 86% of snakebite accidents. Despite extensive evidence of the cytotoxicity of snake venoms, the cellular and molecular mechanisms involved are not fully understood, especially regarding the effects on cell cycle progression and cytoskeleton organization. Traditionally, the effectiveness and quality control tests of venoms and antivenoms are assessed by in vivo assays. Despite this, there is a rising effort to develop surrogate in vitro models according to the 3R principle (Replacement, Reduction, and Refinement). In this study, we treated rat liver cells (BRL-3A) with venoms from five Bothrops species (B. jararaca, B. jararacussu, B. moojeni, B. alternatus, and B. neuwiedi) and analyzed cell viability and IC50 by MTT assay, cell cycle phases distribution by flow cytometry, and morphology and cytoskeleton alterations by immunofluorescence. In addition, we evaluated the correlation between IC50 and the enzymatic and biological activities of each venom. Our results indicated that Bothrops spp. venoms decreased the cell viability of rat liver BRL-3A cells. The rank order of potency was B. jararacussu > B. moojeni > B. alternatus > B. jararaca > B. neuwiedi. The mechanisms of cytotoxicity were related to microtubules and actin network disruption, but not to cell cycle arrest. No clear correlation was found between the IC50 and retrieved literature data of in vitro enzymatic and in vivo biological activities. This work contributed to understanding cellular and molecular mechanisms underlying the Bothrops spp. venom cytotoxicity, which can help to improve envenomation treatment, as well as disclose potential therapeutic properties of snake venoms.
RESUMO
Snake envenomation is classified by the WHO as a neglected tropical disease. Since 2019, due to the 138,000 deaths/year, it has become a target of the organization's plan to reduce deaths and disability. In Brazil, Bothrops jararaca is responsible for 86.8% of accidents, being the subject of many studies to better understand the mechanisms of action of its venom. Toxicity testing commonly involves the use of animal models. Created in 1959, the 3 R’s principle aims to reduce, refine and replace the use of animals with alternative methods with equivalent results. Based on this premise, the objective of this work was to evaluate the toxicity of B. jararaca venom in cell culture as an alternative or complement to toxicity tests performed on animals, aiming at a greater alignment with the 3 R’s principle. RPE-1 cells (15.000 cells/well) were treated with B. jararaca venom between 0.1 μg/mL to 200 μg/mL for 24 h, cell viability and IC50 were calculated by MTT colorimetric assay. Five initial trials established optimal venom concentrations between 10 μg/mL and 200 μg/mL. The results indicated a dose-dependent decrease in cell viability, with a significant drop from 50 μg/mL, presenting viability of 70% and decreasing up to 7% at 200 μg/m. The IC50 calculation resulted in 75.20 μg/mL In conclusion, the work showed that the use of cell culture to analyze cell viability through the MTT test represents an effective method for obtaining data on the cytotoxicity of B. jararaca venom, contributing to the development of alternative methods to the use of animals, according to the principle of the 3 R’s.
O envenenamento por serpentes é classificado pela OMS como uma doença tropical negligenciada. Desde 2019, devido às 138.000 mortes/ano, tornou-se alvo do plano da organização para redução de mortes e invalidez. No Brasil, a espécie Bothrops jararaca é responsável por 86,8% dos acidentes, sendo alvo de muitos estudos para melhor compreensão dos mecanismos de ação de seu veneno. Testes de toxicidade comumente envolvem o uso de modelos animais. Criado em 1959, o princípio dos 3 R’s visa reduzir, refinar e substituir o uso de animais por métodos alternativos com resultados equivalentes. Partindo dessa premissa, o objetivo deste trabalho foi avaliar a toxicidade do veneno de B. jararaca em cultura celular como alternativa ou complemento aos testes de toxicidade realizados em animais, visando um maior alinhamento com o princípio dos 3 R’s. Células RPE-1 (15.000 células/poço) foram tratadas com veneno de B. jararaca entre 0,1 μg/mL a 200 μg/mL por 24 h, a viabilidade celular e o IC50 foram calculados através do ensaio colorimétrico de MTT. Cinco ensaios iniciais estabeleceram as concentrações ideais do veneno entre 10 μg/mL e 200 μg/mL. Os resultados indicaram uma diminuição dose-dependente de viabilidade celular, com queda significativa a partir de 50 μg/mL, apresentando viabilidade de 70% e decaindo até 7% em 200 μg/m. O cálculo do IC50 resultou em 75,20 μg/mL Em conclusão, o trabalho mostrou que o uso da cultura celular para análise da viabilidade celular através do teste de MTT representa um método eficaz na obtenção de dados sobre citotoxicidade do veneno de B. jararaca, contribuindo para o desenvolvimento de métodos alternativos ao uso de animais, segundo o princípio dos 3 R’s.
RESUMO
Understanding the causes and consequences of variation in reproductive strategies is a central theme in studies of avian life history evolution. This study describes the reproductive biology of Synallaxis albescens (Furnariidae) in the cerrado biome of central Brazil. We monitored 35 nests during the 2003 to 2011 breeding seasons, visiting them every 2-4 days. Synallaxis albescens breeds from mid-September to mid-January, builds a retort-shaped nest, and generally lays three immaculate white eggs. Eggs weighed 1.75 g and measured 19.7 by 14.4 mm. Most nests studied were in open cerrado or shrub grassland at an average height above the ground of 0.3 m, with a preference for Davilla elliptica (Dilleniaceae) shrubs as a nesting substrate. Incubation period averaged 18.1 days, while the nestling period averaged 13.6 days. Of 16 closely monitored nests, four were successful (25%), 11 were depredated (69%), and one was abandoned. Predation was similar during incubation (45%) and nestling (55%) phases. In general, the breeding biology of S. albescens was similar to that described previously for this species and for related Furnariidae.
Um tema central em estudos acerca da evolução da história de vida de aves é o entendimento das causas e conseqüências da variação em estratégias reprodutivas. O presente estudo descreve a biologia reprodutiva de Synallaxis albescens (Furnariidae) no bioma cerrado do Brasil central. Nós monitoramos 35 ninhos durantes as estações reprodutivas de 2003 a 2011, visitando-os a cada 2-4 dias. Synallaxis albescens se reproduz da metade de setembro a metade de janeiro, constrói ninho em forma de retorta e geralmente coloca três ovos brancos. Os ovos apresentaram peso de 1,75g e medidas de 19,7 por 14,4 mm. A maior parte dos ninhos estudados estava em Campo sujo e Cerrado ralo a uma altura média, em relação ao solo, de 0.3 m, com uma preferência por arbustos de Davilla elliptica (Dilleniaceae) como substrato para os ninhos. O período de incubação durou em média 18,1 dias, enquanto o período de permanência dos ninhegos durou em média 13,6 dias. Dos 16 ninhos monitorados, quatro obtiveram sucesso (25%), 11 foram predados (69%) e um foi abandonado. A predação foi semelhante durante a fase de incubação (45%) e durante a fase de cuidado dos ninhegos (55%). Em geral, a biologia reprodutiva de S. albescens foi similar ao descrito previamente na literatura para esta espécie, bem como para Furnariidae relacionados.
