Making Waves: New Developments in Toxicology With the Zebrafish

Abstract

The laboratory zebrafish (Danio rerio) is now an accepted model in toxicologic research. The zebrafish model fills a niche between in vitro models and mammalian biomedical models. The developmental characteristics of the small fish are strategically being used by scientists to study topics ranging from high-throughput toxicity screens to toxicity in multi- and transgenerational studies. High-throughput technology has increased the utility of zebrafish embryonic toxicity assays in screening of chemicals and drugs for toxicity or effect. Additionally, advances in behavioral characterization and experimental methodology allow for observation of recognizable phenotypic changes after xenobiotic exposure. Future directions in zebrafish research are predicted to take advantage of CRISPR-Cas9 genome editing methods in creating models of disease and interrogating mechanisms of action with fluorescent reporters or tagged proteins. Zebrafish can also model developmental origins of health and disease and multi- and transgenerational toxicity. The zebrafish has many advantages as a toxicologic model and new methodologies and areas of study continue to expand the usefulness and application of the zebrafish.

Figure 1.

Zebrafish can be used to define and connect changes along the entire spectrum of toxicity research. Zebrafish can be used to model toxicity from the initial intoxication (1), through the molecular initiating event (2), and changes at the cellular (3), tissue (4), and organ (5) levels. Finally, changes in phenotype (6), including alterations in growth and development or behavior and disease outcomes can be observed. In this example, the methylation status of a gene is altered by xenobiotic exposure (2), resulting in altered gene transcription (3), altered neurotransmission (4), and brain dysfunction (5).

Figure 2.

Adult zebrafish behavioral assays. Three common adult zebrafish behavioral assays are the NTT, the LDB, and the OFT. The NTT (A) introduces an adult zebrafish to a novel tank and evaluates endpoints such as time spent in upper and lower zone, latency to zone transitions, and number of zone entries, with an anxious phenotype spending more in the bottom zone. The LDB (B) introduces a zebrafish to a tank set up with dark walls on one half of the tank and white walls on the other half and evaluations similar endpoints such as time spent in light and dark zones, number of zone entries, and the latency between zone entries. The OFT (C) is similar to the rodent test and evaluates thigmotaxis (wall hugging) and exploratory behavior in a novel environment, with anxious fish. Time spent in central versus peripheral zones, latency to zone entry, and number of zone entries, as well as zebrafish startle movements, are recorded. Tracks in each example highlight the swimming pattern of the fish (dot) in the test arena.

Figure 3.

Multi- and transgenerational studies in humans and the zebrafish. Xenobiotic exposure during mammalian pregnancy (A) exposes the mother (F₀), the fetus (F₁), and the primordial germ cells within the fetus (F₂) to potential direct toxic effects. Although the F₁ and F₂ generations have xenobiotic exposure, the F₃ generation is the first generation without exposure, although toxicity may continue through inherited epigenetic toxicity. In zebrafish (B), developmental exposure to a F₀ generation also exposes the germ cells, resulting in indirect xenobiotic exposure to the F₁ generation. The F₂ generation and beyond lack xenobiotic exposure, but may have an altered epigenome. Top blocks represent direct exposure to xenobiotics. The middle blocks represent multigenerational toxicity, where exposure occurred during germ cell stages. The white background represents transgenerational toxicity, where the subjects had no direct xenobiotic exposure and any toxic effects are expected to from epigenetic alterations.