The identification of stress molecules in living fish
Fireflies use light to attract mates and hunt prey; the zebrafish in Dr. Thomas Dickmeis’ laboratory mainly glow in situations of stress. The biologist from the Karlsruhe Institute of Technology (KIT) and two of his doctoral students have created a zebrafish line that can be used as a high-throughput test system for glucocorticoid hormones. Pharmaceutical companies might in future be able to use living vertebrate models, i.e. zebrafish to test new drugs that reduce inflammation and are associated with fewer adverse effects on the organism. The test system would also help scientists to better analyse the effects of certain environmental toxins. In March 2012, the researchers were awarded a prize by the German Society of Endocrinology for their groundbreaking in vivo test system. Dickmeis and his team are also seeking to find ways to enable the zebrafish to glow – or to stop glowing altogether.
Glucocorticoids such as cortisol are steroid hormones that are an integral part of stress response. Higher levels of glucocorticoids prepare us for unpleasant events like confrontations at the workplace, for example. Stress is a complex bodily phenomenon. The experience of stress leads to the release of stress hormones in many organs, resulting in metabolic changes which are associated with the production of glucose in the liver, for example. Such changes can also stop the uptake of glucose in peripheral tissue and improve energy supply to the brain. All this affects the entire organism and prepares us for a potential fight or flight situation. Until now, researchers have mainly used cell cultures to analyse the molecular effects of glucocorticoids, which did not provide them with a greater picture of what is actually happening in an organism.
“Glucocorticoids like cortisol are used in medicine for treating numerous diseases. As stress hormones they can prevent the occurrence of inflammatory reactions of the immune system,” said Dr. Thomas Dickmeis from the Institute of Toxicology and Genetics at the Karlsruhe Institute of Technology (KIT). “However, the chronic uptake of glucocorticoids is associated with many harmful side effects, which is why pharmaceutical companies are constantly looking for new glucocorticoid-like substances that would be better tolerated by patients.” Dickmeis and his team of researchers have developed an in vivo test system with great future potential in the identification of novel glucocorticoids and the screening of compounds for unwanted effects on glucocorticoid signalling.
High-throughput firefly lighting
Dickmeis’ two doctoral students, Meltem Weger and Benjamin Weger, have created a transgenic zebrafish line that glows when exposed to a glucocorticoid. Glucocorticoids normally bind to glucocorticoid receptors in the cell, which then translocate into the nucleus where they are able to switch on different genes. The receptors do this by binding to so-called glucocorticoid response elements (GREs) on the DNA located upstream of these genes. Dickmeis and his team have inserted a firefly luciferase gene downstream of several GREs. The luciferase gene is transcribed when the GREs are activated by the glucocorticoids. The cells in the researchers’ cell line produce a luciferase protein whenever a glucocorticoid is present. When the researchers add firefly luciferin, the luciferase then generates light from this luciferin, resulting in the well-known phenomenon of glowing fireflies. The emission of light proves the presence of a glucocorticoid at an intensity proportional to its dose. “The principle of this quantitative test has been applied for quite some time in cell cultures,” said Dickmeis, adding, “all we have done is to modify the principle for use in a complete living vertebrate.”
The test system involving complete living vertebrates comes with a huge advantage: the immune/stress response of zebrafish is very similar to that of mammals, including humans. In addition, zebrafish breed more easily under laboratory conditions than similar established test systems (e.g. mice) and are able to react to stress just five days after birth. Five-day-old zebrafish are so small that they fit through the tiny openings of microtitre plates, which consist of 96 wells arranged in rows and which enable automated high-throughput experiments to be carried out using state-of-the-art pipetting and microscopy devices. “With our zebrafish mutants we are able to screen substance libraries for compounds with a glucocorticoid-like effect in a relatively short time,” said Dickmeis. “This is perfect for pharmaceutical companies that are looking for new drug candidates.” Dickmeis and his two doctoral students have already shown that their screening system works using a substance library containing a number of known glucocorticoids. They were able to identify the glucocorticoids on the basis of the emitted light. In addition, their system was as accurate as control experiments involving cell cultures.
Global aspects of metabolisation
The importance of using an intact organism as test system should not be underestimated. When they exposed their fish to tributyltin, the researchers highlighted this important advantage. Tributyltin is a highly toxic biocide that does not affect the glucocorticoid signalling system in individual cells. However, the liver of an intact organism that is exposed to tributyltin converts the compound into dibutyltin, which negatively affects the processes happening around glucocorticoids and their receptors. This tributyltin effect would have gone undetected in cell cultures as it is only converted into its effective form in the liver. The chronic exposure of intact organisms to tributyltin can also have negative effects on the metabolic processes in the entire organism.
Researchers nowadays are discussing the phenomenon of “metabolic disruption”, which is similar to the phenomenon of “endocrine disruption” caused by oestrogens in contraceptive pills when they enter the environment and disrupt the hormone balance of animals. “When researchers use cell cultures to test the effect of environmental toxins, they will not notice the global aspects associated with the metabolisation of such toxins,” said Dickmeis going on to add, “our test system helps to detect such effects.” The researchers’ test system also has the potential to be used to analyse the different uptake of toxins or glucocorticoid drugs in the body and in different tissues, or to obtain insights into the degradation processes in different tissues and the resulting chemical intermediary products. This would help pharmaceutical companies to develop drugs that are optimally tailored to such aspects and that would be likely to have fewer harmful side effects than commonly used drugs.
Medical doctors and zebrafish researchers around the world have already expressed their interest in the KIT researchers’ zebrafish line. Meltem Weger and Benjamin Weger were awarded the Schoeller Junkmann Prize by the German Society of Endocrinology (DGE) in March 2012 for the generation of the transgenic zebrafish biosensor that allows the measurement of signalling of glucocorticoid stress hormones in living animals. The innovative test system will also be useful for Dickmeis and his team in the future because the biologist from Karlsruhe has been interested in biological clocks, i.e. the circadian rhythms of human cells, ever since his postdoctoral period. Several years ago, Dickmeis discovered a zebrafish mutant whose cell division activity was not linked to the day-night cycle as is the case with non-mutant animals. “I found that the lack of circadian rhythms is associated with defective glucocorticoid signalling,” said the biologist who now plans to examine his observations further using the luciferase system. He would also like to obtain detailed insights into the relationship between the biological clock, hormones and the metabolism, an area that has so far received scarce attention. Systems biology is the study of biological components, e.g. molecules in individual cells and interacting cells in order to gain insights into the behaviour of entire living systems. How complex will things become when the interactions on the level of entire organisms are also taken into account?
Dr. Thomas Dickmeis
Institute of Toxicology and Genetics (ITG)
Karlsruhe Institute of Technology (KIT)
Tel: +49 (0)721/ 6082 - 65 64
Fax: +49 (0)721/ 6082 - 33 54