Use of fish embryos leads to less animal testing

Stefan Weigt, a researcher of Merck KGaA, Darmstadt, Germany, has developed an improved test with fish embryos that detects interference in embryonic development resulting from drugs or chemicals and avoids animal testing. In recognition of this research, Weigt was presented with a prestigious award in Germany.

The thalidomide scandal was a radical turning point in the development of pharmaceutical products. Between 1958 and 1961, approximately 4,000 children were born with severe deformities in Germany alone, due to the teratogenic effects of the drug. Since then, pharmaceutical products have been rigorously tested for their embryotoxic effects. Until now, this has mostly been done using pregnant rats and rabbits.

However, for several years now, there has also been a test with zebrafish embryos, and Stefan Weigt has made some remarkable improvements to it. In recognition of his efforts, the German federal state of Hesse has presented Weigt with the Animal Welfare Research Award of the State of Hesse. The award honors people and institutions that have made a significant contribution to reducing animal testing or even being able to avoid it completely in some cases. 

What makes zebrafish so suitable for these studies? Several reasons. The molecular timers of embryonic development are very similar in many creatures, including human beings and zebrafish. It is therefore possible to make good predictions about the embryotoxic effect of substances in human beings on the basis of the effect of those same substances on zebrafish embryos. Furthermore, tests with zebrafish embryos, which still live on the reserves in their yolk sacs, are not considered animal tests according to German law. Fish only fall under the provisions of the German Protection of Animals Act (Tierschutzgesetz) after they are capable of independent food intake. Tests with zebrafish embryos are therefore classified as in vitro tests, i.e. as experiments performed in a test tube. They have the same status as cell culture experiments. 

Zebrafish, which are native to India and are three to five centimeters in length, have other advantages too. They spawn almost every week, not just once a year like trout or perch. As a result, embryos are constantly available. They develop outside the mother's body and are almost transparent. The whole course of embryonic development can therefore be monitored under a microscope without having to touch the embryos. "This model is easy to implement," says Weigt. "We can clearly observe any deformities in various tissues during the course of development. In principle, many possible disruptions of normal development can therefore be shown."

What did Stefan Weigt change with respect to the earlier test? The previous one was more complicated, because it was developed with the assumption that zebrafish lack the enzymes to metabolize substances. However, some substances are only embryotoxic when they are changed. In these cases, it is not the initial material but a product of metabolism that damages the embryo. Compounds that exhibit their teratogenic effects only after decomposition — or "metabolic activation" — are called proteratogens. "A few years ago, it was thought that the enzymes for metabolic activation had to be added to the test," says Weigt, who is an animal physiologist. "So testers put individual pieces of rat livers into the test tubes, because the liver has the necessary enzymes. That had certain drawbacks," he continues. "It resulted in an 'indirect need for animals', namely the rats. Furthermore, the addition of these liver fragments was in itself harmful to the embryos."    

That meant the test substances and liver fragments had to be removed from the reaction vessel after only an hour. Because of the brief exposure time, the test was not as meaningful as it might have been otherwise. Many embryotoxic compounds would have had to act longer before their harmful effects were revealed. In addition, the effects on embryonic development were only initially observed for two days instead of three. "Now we're watching the entire process, from the fertilized egg cell to the complete embryo," says Weigt. "We leave the test substance in the reaction vessel the whole time and use physiologically relevant concentrations. This way, we work under conditions that are much more real."    

Using ten well-known sample substances, studied without liver tissue, Weigt showed that zebrafish can be used to test substances whose teratogenic effects only occur after metabolic activation. The typical effects of these substances on embryonic development were clearly demonstrated in zebrafish embryos. Therefore, the zebrafish embryos must have the necessary enzymes. Weigt also studied the embryotoxic effect of the blood thinner warfarin in the zebrafish embryo. Up until then, embryotoxicity studies with various animal species had failed to demonstrate the teratogenic potential of warfarin. But that is precisely what Weigt succeeded in showing for the first time with his improved zebrafish embryo test. 

Today the authorities still require embryotoxicity studies in rats and rabbits prior to granting approval to pharmaceutical products. However, the improved test is already helping to prevent animal testing during clinical drug development, because it can be used as a pretest.