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Mouse-free is the way to be – Nutritional scientists develop a method to replace animal testing

Lab mouse. Photo: Fotolia/Kirill Kurashov.

Lab mouse. Photo: Fotolia/Kirill Kurashov.

It is one of the strongest neurotoxins known to man – and yet is also important for medicine. Botulinum toxin is also known as Botox and is used as an anti-wrinkle treatment. However, before the toxin can be used in aesthetic medicine or for the treatment of diseases, its activity has to be determined in extensive testing. Up until now, this has mostly been done in animal models, specifically in mice. Two existing alternative procedures have been approved for only two specific products and are hence not available for general use. A team led by Prof. Dr. Gerhard Püschel, the head of the Department of Nutritional Biochemistry at the Institute of Nutritional Science, has developed a replacement method that uses isolated nerve cells instead of mice. For this project, the researchers were recently awarded the Research Prize of the state of Berlin.

It takes extreme caution and concentration when researchers conduct experiments with botulinum toxin in the laboratory of the Institute of Nutritional Science. A scientist sits at the sterile work bench wearing a protective suit, gloves, and face mask, and the air in the bench's work space is continuously filtered. He transfers the mixture, which contains only a tiny amount of the toxin, with a pipette into cell cultures. These cultures contain nerve cells impacted by the neurotoxin.

Even tiny amounts of the toxin are lethal

The dose makes the poison – this wisdom applies to botulinum toxin like no other. The substance is not just a highly effective neurotoxin, “it is also an important compound in medicine,” explains Püschel. Botulinum toxin can offer relief for patients suffering from strabismus, migraines, or torticollis.

The bacterium clostridium botulinum produces the poison, for example in spoiled canned food. When the poison enters nerve cells, it inhibits the release of messenger substances. This means that these nerve cells are no longer able to communicate with adjacent muscle cells, thereby paralyzing the muscle. Botulinum poisoning occurs very rarely in Germany, with only about ten cases of botulism reported each year. In most cases the intoxication is caused by consumption of spoiled food. Even a very low dose of the toxin can lead to respiratory paralysis (apnea) and an excruciating death.

Botulium toxin is produced in bacterial cultures for classical medical and aesthetic medical applications. Because it is extremely poisonous and there is no antidote, an overdose must be avoided at all costs. During the production process, part of the toxin is inactivated – yet it is not predictable precisely how much. Because the toxin’s potency varies, the manufacturer has to test every single batch, usually in animal tests. About 150,000 mice are used for this purpose each year in Germany alone and 40,000 of them die of suffocation. “They try to find the concentration at which half of the animals die,” clarifies Püschel.

New test procedure could also work for other toxic substances

Püschel is convinced that the use of animal tests is unnecessary in this case. The two pharmaceutical companies Allergan and Merz, which also sell products with botulinum toxin, have already developed substitute procedures. “Yet these procedures have significant disadvantages,” Püschel asserts. First, the tests are only approved for a specific preparation that is produced by the company, and cannot be applied to other products. Also, the companies will not disclose details of their procedures and thereby prevent broader application and further development. Moreover, these tests are based on immunological processes that require certain antibodies. For cost reasons, these antibodies are produced by injecting tumor cells into the abdomen of mice, which then develop abdominal dropsy (ascites) from which the antibodies are harvested. “It’s actually a cruel irony,” reasons Püschel,"because numerous animals suffer and die unnecessarily from this procedure as well".

Püschel’s team has developed a process for detecting botulinum toxin and assessing its activity, which can probably be applied to a wide range of products containing the neurotoxin. The assay measures the release of a luminescent firefly enzyme. The scientists in his team have genetically modified the enzyme and integrated the DNA into human nerve cell lines. The trick is that the enzyme behaves in nerve cells just like the messenger substances whose release is suppressed by botulinum toxin. The nerve cells release the enzyme together with the messenger substances. Outside the cell the enzyme produces light and is therefore measurable. The greater the activity of the botulinum toxin to which the nerve cells are exposed, the less enzyme is released. “The advantage of the procedure is that it should be applicable for every serotype of the poison,” explains Püschel. The test could therefore deliver reliable results for all possible products that contain the neurotoxin. The scientists found in their first tests that their reporter system can also be used for other substances, such as neurotoxic pesticides. Demand for such a procedure is clear. Gerhard Püschel explains that industry has already expressed interest in the test system.

From an animal reporter system to a human one

Püschel admits that it was purely by chance that his team’s research resulted in a replacement procedure for animal testing. “Originally we wanted to develop a system for our research projects that allowed us to more easily measure nerve cell function.” Püschel learned about the problems with botulinum toxin testing from a newspaper article. He recognized that the approach his team was already investigating would also be appropriate for detecting botulinum toxin, thereby offering a promising new way to make animal testing in this field unnecessary.

After three years of intense work, the laboratory test is now being further refined and will be brought from the bench to the market. The research team wants to establish a model that resembles the natural target as closely as possible. The neuronal tumor cells that have been used up to now will be replaced by human motor neurons produced in cell culture, the botulinum toxin’s natural targets in the human organism. “The cells we’ve used thus far differ in a few ways from motor neurons that are relevant to the effect of botulinum toxin,” says Püschel. While neuronal tumor cells only transmit signals from one nerve cell to another, motor neurons form a direct connection between muscle cells and the nervous system. If the botulinum toxin blocks the release of messenger substances from the motor neurons, this paralyzes the adjacent muscle.

The researchers will now try to use induced pluripotent stem cells (IPS cells) to generate a suitable cell culture and to establish the test. These IPS cells, similar to embryonic stem cells, differentiate to every kind of cell, yet were originally taken from adult human tissue cells and then genetically modified. Püschel’s lab will now test the conditions under which the IPS cells differentiate to the desired motor neurons to which the existing reporter system for detecting botulinum is going to be adapted. This means the researchers will kill two birds with one stone. First, they will establish the best-suited cell type for the botulinum test, and second, they will make the leap from an animal to a human reporter system – which is a significant point for medical research. “If we manage to do this, then our cell culture will have the exact cells that the toxin targets in humans,” says Gerhard Püschel. “And that would be the perfect detection system.”

Püschel knows that black-and-white thinking is not appropriate to judge animal testing. “We also use animal experiments in our research,” he says. “Every time we apply for a permit for this kind of experiment, we have to ponder whether it makes sense and can be justified. That’s required by law.” Before every experiment, researchers have to weigh the significance of the expected results against the pain and suffering caused to the animals in the experiment. And in every single case scientists ask themselves whether there is an alternative. “The knowledge gained and the utility for humans has to be ethically justifiable, considering the animal’s suffering,” says Püschel. “There are no clear or ready answers to the question of what is ethically justifiable. A society has to talk it out. We’re doing that, and that’s good.”


Cell-culture-based in vitro process for determining botulinum toxin activity
Participants: University of Potsdam, Institute for Nutritional Science
Duration: 2012–2015 (SET) and 2016–2019 (EFRE-StaF)
Funding: Foundation for the Promotion of Research on Replacement and Complementary Methods to Reduce Animal Testing (SET), European Fund for Regional Development (EFRE-StaF)

The Researcher

Prof. Dr. Gerhard Püschel studied medicine at the Christian Albrechts University in Kiel and biochemistry at Indiana University in Bloomington (USA). He has been the head of the biochemistry of nutrition department at the University of Potsdam since 1999, and also performs research on mechanisms of insulin resistance in the liver and metabolic disruptions caused by foreign substances.


University of Potsdam 
Institute for Nutritional Science
Arthur-Scheunert-Allee 114–116
14558 Nuthetal
E-Mail: gpuesche@uni-potsdam.nomorespam.de

Text: Heike Kampe
Translation: Dr. Lee Holt/ Brit-Maren Schjeide
Published online by: Agnetha Lang
Contact for the editorial office: onlineredaktion@uni-potsdam.nomorespam.de