Developing the Zebrafish Model for Human Toxicology Research

Robert Tanguay, Ph.D.

June 11, 2018

Robert Tanguay, Ph.D., director of the NIEHS-funded Oregon State University Superfund Research Program Center, has revolutionized the use of Danio rerio, commonly known as zebrafish, for human toxicology research. An innovative approach developed by the Tanguay lab allows scientists to study large numbers of zebrafish during early developmental stages, providing key insights into how chemical exposures in early life affect health.

“When you are at the forefront of a new research field, it can be really intimidating but it can also be really rewarding to create something entirely new,” explained Tanguay.

Creating a new approach

Although zebrafish are now a popular model for studying the impacts of toxic chemicals on human health, they were not widely accepted when Tanguay first began his research in 1996.

According to Tanguay, many researchers at the time felt that zebrafish could provide little insight into the complex functions of human beings. However, in 2013, geneticists found that roughly 82 percent of human genes associated with disease have a match in the zebrafish genome. This high degree of similarity has led to the broad use of zebrafish to study the structural, functional, and behavioral effects of toxic exposures on the human body.

Tanguay has developed an efficient and cost-effective high-throughput screening (HTS) approach which allows his research team to expose and test large numbers of zebrafish at a time. The Tanguay lab has personalized each step of the HTS process for their zebrafish model, a process that took nearly a decade to complete. “Our first step was to figure out how to house this immense volume of fish, which led to the creation of new racks, new instruments, and even new protocols,” explained Tanguay. This new equipment is housed in a 17,000 square foot facility, which Tanguay estimates holds 200,000 adult zebrafish and 10,0000 to 80,000 zebrafish embryos are produced on any given day.

Preparing zebrafish embryos for high-volume testing

While zebrafish are preferred for their rapid development and maturation, this limited time window can potentially complicate research. For this reason, Tanguay and his team developed an automated robotic process to separate the embryos, remove their protective outer shells, and place them into individual micro-wells all within hours of fertilization. Because early life stages are highly similar between species, being able to observe zebrafish as embryos helps researchers to understand how human embryos may react to certain exposures. Getting the most out of this stage is key to understand the developmental impacts of exposure to environmental chemicals.

Once placed into individual wells, Tanguay and his team expose the zebrafish to chemical compounds, such as polycyclic aromatic hydrocarbons (PAHs). Typically, this would be a difficult and time-consuming task, so Tanguay and colleagues developed a barcoding system to expedite the process. Each well is barcoded and logged into a software system that is track each zebrafish throughout their development.

Additionally, an automated instrument performs observation and assessment of any abnormalities that may be present. This data is then transferred to a computer database for analysis.

Applying High-Throughput Screening to PAH Research

The HTS approach developed by Tanguay and his team is particularly useful for researching PAH exposure and its impacts on human health. When inhaled, ingested, or absorbed through the skin, PAHs may cause short-term health effects such as skin irritation, or long-term health issues, such as lung or skin cancer. There are more than 100 different PAHs, and many occur in mixtures, creating endless combinations of PAH mixtures with unknown health effects to humans.

Tanguay and his colleagues want to narrow down which of the hundreds of PAH mixtures are harmful to human health, and at what concentration. In a project led by a Mitra Geier, Ph.D., the Tanguay lab is using HTS to efficiently screen as many of these combinations as possible. Geier used zebrafish and HTS to test over 120 different PAHs at a range of concentrations to determine their dose-toxicity impact. “Using HTS allows us to unleash our imagination on the experimentation process, we are able to test endless combinations of these mixtures for less money and in less time,” said Tanguay.

Translating research for future benefits

Tanguay’s zebrafish research has also been instrumental for industrial manufacturers making decisions on materials. The Tanguay lab screens hundreds of chemicals a year for private manufacturing companies and advises them on potential human health impacts and safer alternatives. “Research translation is the ultimate goal and that is why we take our research a step further into the private sector,” explained Tanguay. He hopes that this type of consultation can prevent industry leaders from using potentially harmful materials in the future.