A research team at the Helmholtz-Centre for Environmental Research (UFZ) has now developed a screening approach based on the zebrafish embryo model that enables high-throughput neurotoxicity testing without the need for conventional animal experiments. In the study, the researchers uncovered the neurotoxic effects and underlying molecular mechanisms of the chemical chlorophene. The study was published in the journal Environmental Health Perspectives.
"To date, only about 200 substances worldwide have been tested for neurotoxic effects through official regulatory studies", says UFZ ecotoxicologist Dr. David Leuthold, lead author of the study. "The reason for this is that these testing procedures are complex, time-consuming, and expensive. In addition, there is the ethical aspect, as these studies are predominantly conducted using rats and mice." What is lacking is a rapid and cost-effective screening method that can quickly and reliably detect neurotoxic effects of chemicals and complex chemical mixtures without the need for conventional animal experiments. And this is precisely where the UFZ study comes in to close this gap.
The UFZ team used the zebrafish embryo model, which is widely applied in toxicological research. One advantage of this model is that around 70 percent of the genes found in the zebrafish (Danio rerio) are also found in humans. The findings from the zebrafish embryo model are therefore likely transferable to humans. Additionally, embryos of the zebrafish are well-suited for high-throughput applications due to their small size and rapid development, providing valuable insights into the function of the nervous system.
Using the zebrafish model, the researchers have designed a screening procedure that allows chemicals to be rapidly tested for neurotoxic effects – including the identification of chemicals that disrupt learning and memory processes. But how can learning and memory be studied in a fish embryo? "We use what is essentially one of the simplest forms of learning: habituation to a re-occurring stimulus," explains Leuthold. "If an acoustic signal is heard, it triggers a startle or escape reflex. However, if it is heard repeatedly, the fish gets used to it and eventually stops responding to the non-threatening stimulus." Alternating between light and dark stimuli also leads to altered swimming behavior in zebrafish embyos. The researchers combined acoustic and visual stimuli in terms of frequency, order, temporal sequence, duration, and intensity, thus designing a defined test procedure.
They initially tested this method using chemical substances whose effects on zebrafish behavior toward visual or acoustic signals were known. "Neurotoxic substances can have very different effects. Some, for example, prevent fish embyos from habituating to an acoustic stimulus, causing their escape reflex to be repeatedly triggered. Other substances can cause habituation to occur much more quickly. Additionally, other visual and acoustic behaviors can be altered," explains Leuthold. "Using these known substances, we were able to generate a kind of behavioral fingerprint, which we can then use to draw conclusions about the way the chemical exposure disrupts nervous system function."
Using their zebrafish platform, the researchers then tested ten selected substances known to influence a receptor system (NMDAR), which plays a special role in learning and memory. Whether they also exhibit neurotoxic effects in zebrafish was not yet known. "With our screening approach, we were able to demonstrate significant effects on learning behavior for six substances. They therefore clearly exhibited a neuroactive effect," says Leuthold. One substance in particular caught the researchers' attention: Chlorophene, a chemical belonging to the group of biocides. Unlike the other substances, chlorophene did not lead to faster habituation to acoustic stimuli, but instead blocked learning behavior entirely. And another interesting finding emerged: Under the influence of chlorophen, the fish embryos still responded to acoustic stimuli, but not to visual ones. "This phenomenon is called paradoxical excitation and occurs with certain narcotics," says Leuthold. It was previously unknown that chlorophene could also have this effect, which is why the researchers wanted to further investigate the underlying mechanism of action.
They came across a study by US-American colleagues in which narcotics were tested in the zebrafish model. The study showed that paradoxical excitation can be mediated by specific receptors (GABAA), which play an important role in our central nervous system and are pivotal for controlling behavior. Could the effect of chlorophene be inhibited if GABAA receptors are blocked? Leuthold: "When GABAA receptors were blocked, the fish embryos exposed to chlorophene recovered their ability to respond to visual stimuli. However, blocking the receptors could not reverse the altered learning behavior. We learned that chlorophene has multiple molecular mechanisms."
But first, the researchers wanted to validate their hypothesis regarding chlorophene's mechanism of action via GABAA receptors through further tests. For this, they used neurons isolated from mice and human neuronal cell models. In collaboration with colleagues from the University of Leipzig and the Leibniz Institute for Environmental Medicine in Düsseldorf, the researchers were able to demonstrate that chlorophene also acts via GABAA receptors. Computer models that match the chemical structure with possible receptors also predicted binding to GABAA receptors. Chlorophene's mechanism of action via GABAA receptors was thus proven.
But what about chlorophene's other mechanism of action, which alters learning behavior? Could the first-mentioned NMDA receptor system be behind this? Further studies indicated that chlorophene probably doesn't interact directly with the receptor. In the US study with the narcotics tested in the zebrafish model, the researchers found evidence of another receptor system that could play a role in paradoxical excitation: certain potassium channels. "So we came up with the idea of testing the painkiller flupirtine, which acts via these potassium channels, in our zebrafish platform," explains Leuthold. "And indeed – flupirtine elicited almost the same behavioral patterns as chlorophene, including reduced learning behavior! Chlorophene presumably acts in a very similar, if not identical, way via these potassium channels."
The researchers hope that their screening approach will help enable chemicals and chemical mixtures to be tested for neurotoxic effects on a large scale – quickly, cost-effectively, and without the need for conventional animal testing – so that risks to humans and the environment can be identified at an early stage. "Our zebrafish platform is in line with the EU Chemicals Strategy and the concept of the European Green Deal, as it can identify hazardous chemicals early, before they cause harm," says Leuthold.
Prof Dr Tamara Tal, who heads the working group at the UFZ where the study was conducted, emphasises: "Regulatory authorities are generally skeptical about using toxicity data generated in zebrafish to make chemical regulations for humans. When we demonstrate that the way these chemicals disrupt how the brain develops and functions is specifically conserved in zebrafish, mouse, and human models, we build confidence in the use of zebrafish behavior-based data to fill the vast void in neurotoxicity testing to ultimately improve human health from the harmful effects of neurotoxic chemicals.”
Publication
David Leuthold, Nadia K. Herold, Jana Nerlich, Kristina Bartmann, Ilka Scharkin, Stefan J. Hallermann, Nicole Schweiger, Ellen Fritsche, Tamara Tal: Multi-behavioral phenotyping in early-life-stage zebrafish for identifying disruptors of non-associative learning; Environmental Health Perspectives, https://doi.org/10.1289/EHP16568
Further information:
Accelerated Testing of More Substances – Towards Better Chemicals Regulation
Further information
Dr David Leuthold
UFZ Department Ecotoxikology
david.leuthold@ufz.de
Prof Dr Tamara Tal
UFZ Department Ecotoxikology / Head of Working Group Mechanistic Toxicology
tamara.tal@ufz.de