Alternative methods in toxicity testing: the current approach

Alternative methods are being developed to reduce, refine, and replace (3Rs) animals used in experiments, aimed at protecting animal welfare. The present study reports alternative tests which are based on the principles of the 3Rs and the efforts made to validate these tests. In Europe, several methodologies have already been implemented, such as tests of irritability, cell viability, and phototoxicity as well as in vitro mathematical models together with the use of in silico tools. This is a complex process that spans from development to regulatory approval and subsequent adoption by various official entities. Within this regulatory framework is REACH, the European Community Regulation for chemicals and their safe use. In Brazil, the BraCVAM (Brazilian Center for the Validation of Alternative Methods) was recently established to validate alternative methods and stimulate incorporation of new methodologies. A new vision of toxicology is emerging for the 21st century (Tox-21), and the subsequent changes are shaping a new paradigm.

Métodos alternativos estão sendo desenvolvidos para a redução, o refinamento e a substituição (3R) do número de animais utilizados em experimentos, visando ao seu bem-estar. Esses testes alternativos baseiam-se no princípio dos 3R e esforços têm sido empregados para que sejam validados. Na Europa, diversas metodologias já foram implantadas tais como: testes de irritabilidade, testes de viabilidade celular, testes de fototoxicidade e modelos matemáticos in vitro, além do uso de ferramentas in silico. Esse é um processo complexo, que abrange desde o seu desenvolvimento até a aceitação regulatória e posterior adoção por diversas organizações oficiais. No contexto regulatório está o REACH, o Regulamento da Comunidade Européia, para produtos químicos e sua utilização segura. No Brasil, o BraCVAM (Centro Brasileiro de Validação de Métodos Alternativos) foi recentemente estabelecido para validação de métodos alternativos e estímulo à incorporação de novas metodologias. Uma nova visão de toxicologia vem surgindo para o século XXI (Tox-21) e as mudanças ocasionadas promoverão um novo paradigma.

Manejo general del intoxicado / General management of intoxicated

Intoxications have become a relevant complaint in the emergency room since the second half of the last century. Toxidromes have been replaced by a more practical combined analysis of vital signs, directed physical examination and selected laboratory tests. Most of the mortality can be prevented by the correct and opportune implementation of general management strategies, including supportive care, prevention of absortion, enhancement of elimination and extracorporeal removal of toxins. Through the following pages we will review many of different diagnostic and therapeutic alternatives...

Characterization of a Mutant Bacillus thuringiensis d-endotoxin with enhanced stability and toxicity / Caracterización de una delta endotoxina mutante de Bacillus thuringiensis con estabilidad y toxicidad aumentadas

The centrally located a-helix 5 of Bacillus thuringiensis d-endotoxins is critical for insect toxicity through ion-channel formation. We analyzed the role of the highly conserved residue Histidine 168 (H168) using molecular biology, electrophysiology and biophysical techniques. Toxin H168R was ~3-fold more toxic than the wild type (wt) protein whereas H168Q was 3 times less toxic against Manduca sexta. Spectroscopic analysis revealed that the H168Q and H168R mutations did not produce gross structural alterations, and that H168R (Tm= 59 °C) was more stable than H168Q (Tm= 57.5 °C) or than the wt (Tm= 56 °C) toxins. These three toxins had similar binding affinities for larval midgut vesicles (Kcom) suggesting that the differences in toxicity did not result from changes in initial receptor binding. Dissociation binding assays and voltage clamping analysis suggest that the reduced toxicity of the H168Q toxin may result from reduced insertion and/or ion channel formation. In contrast, the H168R toxin had a greater inhibition of the short circuit current than the wt toxin and an increased rate of irreversible binding (kobs), consistent with its lower LC50 value. Molecular modeling analysis suggested that both the H168Q and H168R toxins could form additional hydrogen bonds that could account for their greater thermal stability. In addition to this, it is likely that H168R has an extra positive charge exposed to the surface which could increase its rate of insertion into susceptible membranes.

La a-Hélice 5 del domino I de las d-endotoxinas de Bacillus thuringiensis, es crítica para la toxicidad de las toxinas contra insectos al participar en la formación de canales iónicos. La participación en la función tóxica del residuo Histidina 168 (H168) –el cual es altamente conservado– fue estudiada mediante técnicas de biología molecular, electrofisiología y biofísica. La toxina mutante H168R fue ~ 3 veces más tóxica que la toxina silvestre (ts) en Manduca sexta, mientras que H168Q fue 3 veces menos tóxica. Los análisis espectroscópicos indicaron que las mutaciones no producen alteraciones estructurales significativas y que la toxina H168R (Tm= 59 °C) es más estable que las toxinas H168Q (Tm= 57.5 °C) y wt (Tm= 56 °C). Las tres toxinas exhibieron uniones de afinidad similares (Kcom) en vesículas de intestino de larvas de insecto, indicando que las diferencias en la toxicidad no se deben a cambios en la unión inicial al receptor. Los ensayos de unión/disociación y fijación de voltaje mostraron que la reducción de la toxicidad de la toxina H168Q se puede atribuir a una disminución en la inserción y/o en la formación de canales iónicos. De otro lado, H168R mostró una inhibición a la corriente de corto circuito mayor que la ts y un aumento en unión irreversible (kobs), lo cual es consistente con un menor valor de CL50. La modelación molecular sugiere que H168Q y H168R forman puentes de hidrógeno adicionales, lo que les confiere mayor estabilidad térmica. Adicionalmente, es probable que H168R tenga una carga positiva extra expuesta en la superficie, lo cual aumentaría su tasa de inserción en membranas susceptibles.