The zebrafish embryo has become an important vertebrate model for assessing drug effects. It is well suited for studies in genetics, embryology, development, and cell biology. Zebrafish embryos exhibit unique characteristics, including ease of maintenance and drug administration, short reproductive cycle, and transparency that permits visual assessment of developing cells and organs. Because of these advantages, zebrafish bioassays are cheaper and faster than mouse assays, and are suitable for large scale drug screening. Here we describe the use of zebrafish bioassays for assessing toxicity, angiogenesis, and apoptosis. Using 18 chemicals, we demonstrated that toxic response, teratogenic effects, and LC50 in zebrafish are comparable to results in mice. The effects of compounds on various organs including the heart, brain, intestine, pancreas, cartilage, liver, and kidney were observed in the transparent animals without complicated processing, demonstrating the efficiency of toxicity assays using zebrafish embryos. Using endogenous alkaline phosphatase staining and a whole-animal enzyme assay, we demonstrated that SU5416 and flavopiridol, compounds shown to have anti-angiogenic effects in mammals, inhibit blood vessel growth in zebrafish, and the bioassay is suitable for high throughput screening. We also demonstrated that in vivo acridine orange staining can be used to visualize apoptotic events in embryos treated with brefeldin A, neomycin, or caspase inhibitors. After in vivo staining, acridine orange can be extracted and quantitated using a fluorescence microplate reader, providing a screening system for agents that modulate apoptosis.

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The increasing number and diversity of compounds made available by rapid synthesis techniques such as combinatorial chemistry, combined with high throughput or ultra-high throughput in vitro drug screening assays, generate large number of preliminary "hits." However, validating these preliminary hits by mammalian animal models is very slow and costly, resulting in a gap in the drug development process. The zebrafish is a vertebrate model organism that can bridge this gap. Zebrafish-based assays combine the advantages of higher throughput analysis (compared to mammalian models) and higher relevance to humans (compared to in vitro and invertebrate models). Drug screening assays in diverse formats have been developed using zebrafish, including visual assessment of effects on organs in the transparent embryo and quantitative assays using microplates. The microplate-based quantitative assays can be performed in a similar fashion as cell based assays, and can be used for primary drug screening at high throughput. In this article, we review the recent advance in applying in vivo zebrafish assays for testing drug toxicity and drug effects on angiogenesis and apoptosis.

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Currently, in vivo assays are usually performed at or after lead optimization stage. This is partly due to the low speed and high cost of the conventional animal models (typically rodents) and the relatively high number of preliminary hits from HTS. With alternative small animal models entering drug discovery research, it is now possible to perform in vivo testing earlier in the process. Thus, researchers have developed model systems using both invertebrates and vertebrates for drug screening. The small size, high fecundity, and experimental tractability of these animals enable cost-effective and rapid screening of numerous compounds.

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