title: Set-up of an infrared fast behavioral assay using zebrafish (Danio rerio) larvae, and its application in compound biotoxicity screening.
abstract: Zebrafish (Danio rerio) is increasingly employed for evaluating toxicity and drug discovery assays. Commonly experimental approaches for biotoxicity assessment are based on visual inspection or video recording. However, these techniques are limited for large-scale assays, as they demand either a time-consuming detailed inspection of the animals or intensive computing resources in order to analyze a considerable amount of screenshots. Recently, we have developed a simple methodology for tracking the locomotor activity of small animals cultured in microtiter plates. In this work, we implemented this automatic methodology, based on infrared (IR) microbeam scattering, for measuring behavioral activity in zebrafish larvae. We determined the appropriate culture conditions, number of animals and stage of development to get robust results. Furthermore, we validated this methodology as a rapid test for evaluating toxicity. By measuring the effects of reference compounds on larvae activity, we were able to estimate the concentration that could cause a 50% decrease in activity events values (AEC₅₀), showing a strong linear correlation (R²  = 0.91) with the LC₅₀ values obtained with the standard DarT test. The toxicity order of the measured compounds was CuSO4  > 2,4-dinitrophenol > 3,4-dichloroaniline > SDS > sodium benzoate > EDTA > K₂CrO4 ; regarding solvents, EtOH ≈ DMSO. In this study, we demonstrate that global swimming behavior could be a simple readout for toxicity, easy to scale-up in automated experiments. This approach is potentially applicable for fast ecotoxicity assays and whole-organism high-throughput compound screening, reducing the time and money required to evaluate unknown samples and to identify leading pharmaceutical compounds.
authors: Fundación Instituto Leloir, CONICET, Buenos Aires, Argentina; Instituto de Biología Molecular y Celular de Rosario, CONICET; and Facultad de Ciencias Bioquímicas y Farmacéuticas, Universidad Nacional de Rosario, Argentina.
authors: Darío
authors: D
authors: Bichara
authors: Nora B
authors: NB
authors: Calcaterra
authors: Silvia
authors: S
authors: Arranz
authors: Pablo
authors: P
authors: Armas
authors: Sergio H
authors: SH
authors: Simonetta
mesh_terms: D019297:2,4-Dinitrophenol / Q000633:toxicity; D000814:Aniline Compounds; D000818:Animals; D002840:Chromates / Q000633:toxicity; D019327:Copper Sulfate / Q000633:toxicity; D004305:Dose-Response Relationship, Drug; D054750:Ecotoxicology / Q000379:methods*; D004492:Edetic Acid / Q000633:toxicity; D005260:Female; D007814:Larva / Q000187:drug effects; D007928:Lethal Dose 50; D008297:Male; D009043:Motor Activity / Q000187:drug effects*; D017680:Potassium Compounds / Q000633:toxicity; D015203:Reproducibility of Results; D012542:Scattering, Radiation; D020160:Sodium Benzoate / Q000633:toxicity; D012967:Sodium Dodecyl Sulfate / Q000633:toxicity; D018675:Toxicity Tests; D015027:Zebrafish
chemical_list: D000814:Aniline Compounds; D002840:Chromates; D017680:Potassium Compounds; C014464:3,4-dichloroaniline; D012967:Sodium Dodecyl Sulfate; C027373:potassium chromate(VI); D004492:Edetic Acid; D019327:Copper Sulfate; D020160:Sodium Benzoate; D019297:2,4-Dinitrophenol
keywords: Danio rerio; automated assay; behavior; biotoxicity; high throughput screening; tracking method; zebrafish