ToxSci Advance Access published online on March 28, 2008
Toxicological Sciences, doi:10.1093/toxsci/kfn065
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Development of a new screening assay to identify proteratogenic substances using zebrafish Danio rerio embryo combined with an exogenous mammalian metabolic activation system (mDarT)
* Institute of Toxicology, Merck KGaA, 64293 Darmstadt, Germany
Institute of Hydrobiology, TU Dresden, 01062 Dresden, Germany
Corresponding author address: Thomas H. Broschard, PhD, Merck KgaA, Institute of Toxicology, Building U9, Frankfurter Landstr. 250, 64293 Darmstadt Germany, Tel: +49(0)6151 72 2173, Fax: +49(0)6151 72 912173, Email: thomas.broschard{at}merck.de
Received February 6, 2008; revision received March 14, 2008; accepted March 14, 2008
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Abstract |
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The assessment of teratogenic effects of chemicals is generally performed using in vivo teratogenicity assays e.g., in rats or rabbits. We have developed an in vitro teratogenicity assay using the zebrafish Danio rerio embryo combined with an exogenous mammalian metabolic activation system (MAS), able to biotransform proteratogenic compounds. Cyclophosphamide (CPA) and ethanol were used as proteratogens to test the efficiency of this assay. Briefly, the zebrafish embryos were co-cultured at 2 hpf (hours post fertilization) with the test material at varying concentrations, induced male rat liver microsomes and NADPH for 60 min at 32°C under moderate agitation in Tris-buffer. The negative control (test material alone) and the MAS control (MAS alone) were incubated in parallel. For each test group, 20 eggs were used for statistical robustness. Afterwards fish embryos were transferred individually into 24-well plates filled with fish medium for 48 hours at 26°C with a 12 hour-light cycle. Teratogenicity was scored after 24 and 48 hpf using morphological endpoints. No teratogenic effects were observed in fish embryos exposed to the proteratogens alone, i.e. without metabolic activation. In contrast, CPA and ethanol induced abnormalities in fish embryos when co-incubated with microsomes. The severity of malformations increased with increasing concentrations of the proteratogens. We conclude that the application of microsomes will improve and refine the Danio rerio teratogenicity assay as a predictive and valuable alternative method to screen teratogenic substances.
Key Words: zebrafish; cyclophosphamide; ethanol; alternatives to animal testing; teratogenicity; metabolic activation.
Authors e-mail: francois.busquet{at}merck.de, friedrich.vonlandenberg{at}merck.de, roland.nagel{at}tu-dresden.de, stefan.o.mueller{at}merck.de, nicole.huebler{at}merck.de, thomas.broschard{at}merck.de