Zebrafish study investigates how exposure to PFAS affects brain development

Due to their special properties – heat resistance, water and grease repellency and high durability – PFAS are used in many everyday products (e.g. cosmetics, outdoor clothing and coated cookware). But it is precisely these qualities that make them so problematic.

“Because some PFAS are chemically stable, they accumulate in the environment and enter our bodies through the air, drinking water and food,” says UFZ toxicologist Prof. Dr. Tamara Tal. Even with careful consumption, it is virtually impossible to avoid this group of substances, which has been produced since the 1950s and now includes thousands of different compounds.

“There is a great need for research, especially when it comes to developing rapid, reliable and cost-effective testing systems for assessing the risks of exposure to PFAS,” says Tal. So far, the consequences for the environment and health are difficult to estimate.

In their current study, the researchers examined how exposure to PFAS affects brain development. For this they used the zebrafish model, which is frequently used in toxicological research. The study is published in the news Environmental health perspectives.

An advantage of this model is that approximately 70% of the genes that occur in zebrafish (Danio rerio) also occur in humans. The findings from the zebrafish model can therefore probably be transferred to humans. In their experiments, the researchers exposed the zebrafish to two substances from the PFAS group (PFOS and PFHxS), which have a similar structure.

The researchers then used molecular biological and bioinformatic methods to investigate which genes were disrupted in the brains of fish larvae exposed to PFAS compared to control fish that had not been exposed.

“In the zebrafish exposed to PFAS, the peroxisome proliferator-activated receptor (ppar) gene group, which is also present in a slightly modified form in humans, was particularly active,” says Sebastian Gutsfeld, Ph.D. student at UFZ and first author of the study. “Toxicity studies have shown that this is the case as a result of exposure to PFAS, albeit in the liver. We have now been able to demonstrate this for the brain as well.”

But what consequences does altered activity of the ppar genes caused by exposure to PFAS have for the brain development and behavior of zebrafish larvae? The researchers investigated this in further studies with the zebrafish model. They used the CRISPR/Cas9 method, also known as gene scissors.

“Using genetic scissors, we could selectively cut individual or multiple ppar genes and prevent them from functioning normally,” Gutsfeld explains. “We wanted to find out which ppar genes are directly linked to a change in larval behavior caused by PFAS exposure.”

Proof of the underlying mechanism was provided immediately. Unlike genetically unaltered zebrafish, gene scissor knockdown fish should show no behavioral changes after exposure to PFAS.

The two behavioral endpoints

In one series of experiments, the researchers continuously exposed the zebrafish to PFOS or PFHxS during their early development phase between day one and day four, and in another series of experiments only on day five. On the fifth day, the researchers then observed swimming behavior. For this they used two different behavioral endpoints.

In one endpoint, swimming activity was measured during a prolonged dark phase. PFAS-exposed fish swam more than fish not exposed to PFAS, regardless of whether they were exposed to PFAS continuously during brain development or shortly before the behavioral test. Interestingly, hyperactivity was only present when the chemical was nearby. When the researchers removed PFOS or PFHxS, the hyperactivity decreased. In the second endpoint, the startle response after a dark stimulus was measured.

“In zebrafish exposed to PFOS for four days, we observed hyperactive swimming behavior in response to the stimulus,” says Gutsfeld. In contrast, zebrafish exposed to PFOS or PFHxS only on the fifth day did not show a hyperactive startle response.

Based on these responses, the researchers conclude that exposure to PFOS is associated with abnormal consequences, especially during sensitive phases of brain development. Using knockdown zebrafish, the researchers identified two genes from the ppar group that mediate the behavior induced by PFOS.

“Because these genes are also present in humans, it is possible that PFAS also have similar effects in humans,” concludes Tal. The scientists working with Tal want to investigate the neuroactive effects of other PFAS in future research projects and expand the method so that it can ultimately be used to assess the risk of chemicals in the environment, including PFAS.