Zebrafish assay for apoptosis answers HTS call

Apoptosis, the genetically programmed process of precise, predictable cell suicide, occurs as a normal physiological process for removing unnecessary cells throughout the life of an organism. Inappropriate apoptosis has been shown to be the cause of many diseases, and there has been a lot of work on ways to stimulate the process in diseases such as cancer.

Phylonix has developed a quantitative, dye-based assay that uses an automated liquid handling workstation and microplate reader to monitor apoptotic cells in live zebrafish. These can be visualised in the fish using a simple dye (acridine orange) and with no need for complicated processing, according to the company.

Chuenlei Parng, the principal investigator in the research, said that conventional cell-based assays evaluate the potential effects of drugs in culture, but cannot assess the complex metabolism that affects actual drug efficacy or causes toxicity in animals and humans. Therefore, many drugs that appear effective in cell-based assays fail in subsequent, costly animal testing.

In contrast, the zebrafish has several important advantages for drug screening. They are small, inexpensive to maintain and easily bred in large numbers; eggs are externally fertilised and a single mating produces 100-200 eggs.

Single embryos can be maintained in fluid volumes as small as 100 microlitres for the first six days of development and cultured in microtitre wells. Chemicals can then be added directly to the fish water, permeating the intact embryo. Standard microtitre plate readers can be used for quantitation, making this format particularly attractive for high throughput drug screening.

"This convenient animal model will serve as an intermediate step between cell-based evaluation and animal testing for drug evaluation,"​ said Parng.

Development of agents that modulate apoptosis is a major focus of biopharmaceutical research and could permit therapeutic intervention in not only cancer but also heart disease, stroke, AIDS, autoimmunity, and degenerative diseases.

Phylonix is developing a family of zebrafish microplate assays with claimed advantages such as short assay time, single dosing, a small amount of drug required for each test and low cost. The estimated cost of each zebrafish assay is less than $100.

Whole embryo testing has previously been performed on invertebrates, including fruitfly and nematode, however, these organisms are not closely related to humans and they lack many of the same organs and enzymes. Therefore, their use as comparative models for toxicity effects in humans is limited, claims Phylonix.

In contrast, all essential components of vertebrate form and organ development are mimicked in the transparent zebrafish and their molecular basis is either identical or similar, underscoring the potential for use in research on human diseases.