A Brief History of Zebrafish
The zebrafish is a freshwater about 4 centimeters in length fish with a natural habitat from the clear waters of Ganges and Brahmaputra River basins. The fish was initially in the 1960’s likely employed as a model of choice due to its transparent nature; read outs could therefore be made in adults or offspring through mere visual observations. The model was employed primarily to understand development biology by several groups such as Hisaoka KK, Abedi ZH, Weis JS, Schmatolla E, Gilbert W, Eisen JS, Krauss S, Grunwald DJ, Streisinger G, Ross LS, Parrett T and Weinberg ES. However, with the development of methods to induce mutation, the zebrafish became an attractive model. With the total time span of few days to visualizing loss of function due to mutation in the whole animal, the zebrafish fish was becoming a treasure trove to uncover molecular structure and function relationships. By 2013, the Wellcome Trust Sanger Institute a non-profit British genomics and genetics research institute, sequenced the zebrafish genome. They had identified that 70% of the zebrafish gene share similar gene with humans. This catapulted the fish from being a mere information provider on structure, function & mechanisms to a deeper use in toxicology and pharmacology. The fish is now widely used in predictive toxicology, efficacy screening and pharmacology aspects across all therapeutic portfolios.
Guidelines on the use of Zebrafish for Pre-Clinical Development
The feedback from FDA on the use of zebrafish for Safety Pharmacology & Efficacy is that the zebrafish, should be capable of detecting the same endpoints with the same specificity and sensitivity as provided by traditional methods. Therefore, current lab practices employ the same primary endpoints in zebrafish which have been employed in larger animals or better adopted from clinical trials. In certain cases, the zebrafish testing labs have turned this a notch higher by adopting the use of humanized zebrafish (xenotransplant of human cells or human gene inserts) for several therapeutic areas including cancer and neurodegenerative disorders. Overall, the zebrafish has become a valuable and quicker in vivo screening platform that provides data equivalence to human and large animal studies that can be employed across all therapeutic areas. The literature of relevance data generated by zebrafish is growing year on year with about 2000 peer reviewed publications made every year.
In the zebrafish embryo toxicity, the freshly fertilized embryos are exposed to the test item in 5 concentrations over a course of 96 hours. Every 24 hours, four apical parameters of acute toxicity are recorded, and the lethal concentration (LC50) is calculated based on the 96 hours observation.
ICH 14 (FDA, 2005)
It is required that any drug candidate prior to human evaluation is tested in vitro for its effect on hERG channel expressed in cell lines and in vivo for QT prolongation. The availability of a zebrafish model with the ability to reveal QT interval-prolonging effects of compounds through multiple mechanisms could be employed.
ICH 2005; ICH 2015
Each drug in development for administration to women of childbearing potential must be tested for developmental toxicity in rodent and non-rodent species. Zebrafish could be employed
CNS studies including behavior, learning and memory, neurochemistry, optomotor, and/or electrophysiology examinations are recommended before product approval. Zebrafish could be employed.
This guidance document does allow testing laboratories to use new and innovative biocompatibility testing methods. The zebrafish embryo toxicity (ZET) test employs the embryos of the zebrafish (Danio rerio) to assess the toxicity of medical device polymers, drug-device combination products, and other medical device leachates.
This International Standard specifies a semi-static method for determination of toxicity of chemicals, waters and wastewaters to embryos and early larval developmental stages of a species of freshwater fish, Where necessary this determination may include an acute toxicity test using Danio rerio to determine the 96-h LC50 for zebrafish in accordance with ISO 7346-1, ISO 7346-2 or ISO 7346-3.
Examples of approved compounds by therapeutic segments and key findings in zebrafish with relevance to Pharmacological Efficacy
Margiotta-Casaluci L, Owen SF, Rand-Weaver M, Winter MJ. Testing the Translational Power of the Zebrafish: An Interspecies Analysis of Responses to Cardiovascular Drugs. Front Pharmacol. 2019;10:893. Published 2019 Aug 16. doi:10.3389/fphar.2019.00893
Microtubule arrays of the zebrafish yolk cell: organization and function during epiboly. L. Solnica-Krezel, W. Driever. Development 1994 120: 2443-2455
Carvalho FR, Fernandes AR, Cancela ML, Gavaia PJ. Improved regeneration and de novo bone formation in a diabetic zebrafish model treated with paricalcitol and cinacalcet. Wound Repair Regen. 2017 May;25(3):432-442. doi: 10.1111/wrr.12536. Epub 2017 Apr 27. PMID: 28380670.
Adams KN, Takaki K, Connolly LE, et al. Drug tolerance in replicating mycobacteria mediated by a macrophage-induced efflux mechanism [published correction appears in Cell. 2011 Apr 1;145(1):159]. Cell. 2011;145(1):39-53. doi:10.1016/j.cell.2011.02.022
Zhou J, Guo SY, Zhang Y, Li CQ. Human prokinetic drugs promote gastrointestinal motility in zebrafish. Neurogastroenterol Motil. 2014 Apr;26(4):589-95. doi: 10.1111/nmo.12306. Epub 2014 Feb 7. PMID: 24533865
Nam YH, Moon HW, Lee YR, et al. Panax ginseng (Korea Red Ginseng) repairs diabetic sensorineural damage through promotion of the nerve growth factor pathway in diabetic zebrafish. J Ginseng Res. 2019;43(2):272-281. doi:10.1016/j.jgr.2018.02.006
Zaig S, Scarpellini C, Montandon G. Respiratory depression and analgesia by opioid drugs in freely-behaving larval zebrafish. bioRxiv; 2020. DOI: 10.1101/2020.09.30.320267.
Effects of vitamin K deficiency and its mechanisms in vertebrate's early development: zebrafish as a model. Granadeiro, Luís Carlos Serrachino. Disertação de mestrado, Ciências Biomédicas, Departamento de Ciências Biomédicas e Medicina, Universidade do Algarve, 2015. http://hdl.handle.net/10400.1/7717
Toxicity Assessements that can be performed in Zebrafish