Our Tiny Allies in Toxicological Testing - Zebrafish and their Embryos as Alternative Models

By Aaradhya Diwan | December 19th, 2024

The fields of pharmacology and toxicology have grown considerably in the past 20 years, with rapid advances in the development and usage of new pharmaceuticals and chemicals aimed at improving human health and well-being. Alongside the growth of this scientific discipline, as researchers understand the effects of these chemicals (both intended and off-target) in more detail, the need for recognizing and studying potentially toxic substances in the environment has arisen. As these chemicals pollute the air, water, and soil, there is an ever-increasing risk of poisoning not just the environment, but also human health. To prevent the release and unchecked harm that such substances might cause, governments and institutions around the world have brought into play the field of environmental toxicology, which assesses substances to determine their impact on human and ecosystem health and how these substances should be regulated.

When depicted in such a way, toxicology exists as a crucial process to save our Earth from the negative impacts of unrestrained anthropogenic development. However, a large portion of toxicological tests are conducted on animals (specifically, vertebrates) in order to assess the biological and environmental endpoints that are needed to set regulatory limits. The animal testing of these chemicals has become so ossified that we do not question it from an ethical perspective, and we hesitate to consider reasonable alternative models that research labs and toxicology centers can use in their place.

New Approach Methodologies (NAMs for short), incorporated into the Toxic Substances Control Act and employed by the Environmental Protection Agency’s Strategic Plan to Promote the Development and Implementation of Alternative Test Methods in 2018, are methods and technologies that aim to Replace, Reduce, and Refine (3R-Principle) the usage of vertebrate animal models in testing. On the current list of NAMs as updated in 2021, there are 32 alternative tests that can be used to determine adverse effects on human systems and 19 more tests for biotic/ecological systems, as validated by the Organization for Economic Co-operation and Development (OECD) and the Interagency Coordinating Committee on the Validation of Alternative Methods (ICCVAM).

This article will explore the zebrafish, Danio rerio, as a specific animal model and discuss whether it currently serves its potential role as a NAM. The article will show that these models offer powerful insight for detecting the negative impacts of chemical substances on ecosystem health and human health.

The Zebrafish Model

The zebrafish is a popular model for clinical research as it can offer insight into human health effects of medications and drugs. Zebrafish are easily maintained with low costs; they rapidly develop from embryo to adult; they produce a large number of offspring per reproductive cycle; their genome has been entirely sequenced so they are useful for genetic testing; their embryos and a few strains of zebrafish are transparent so physical effects can be studied; and they have specific similarities to the human system (such as developed cardiovascular and nervous systems).

The zebrafish have 3 general stages of development - the embryo, the larva, and the adult. The adult zebrafish is a vertebrate animal model, so toxicology testing with the adult itself is a target of substitution by the appropriate NAMs. Here lies an idiosyncrasy with zebrafish as an ‘animal’, where animal has been officially defined (by the Animal & Plant Health Inspection Service (APHIS) of the US Department of Agriculture) by its status as a vertebrate. So, do the embryos and larvae count as animals? And how is oversight of zebrafish managed in this dilemma under the Public Health Service Policy on Humane Care and Use of Laboratory Animals by the Office of Laboratory Animal Welfare (OLAW)? What has been decided and incorporated into IACUC protocols by research labs back in 2009 when the question first arose, is that 8 days post fertilization (dpf) of the embryos is necessary to track them under animal welfare services and regulation. At the time of 8 dpf, the zebrafish have entered the mid larval development stage, where they have developed a spine, are able to freely swim, and can legally feel pain under Category C of the USDA Pain & Distress Categories.

There are already two tests accepted by the EPA and listed in the NAM Work Plan where zebrafish embryos are used as NAMs - they are OECD 212 [Short-term Toxicity Test on Embryo and Sac-fry Stages] and OECD 236 [Fish Embryo Acute Toxicity (FET) Test]. These test guidelines consist of a protocol where the embryos and early (immobile) larvae are exposed to varying concentrations of the chemical to determine the acute toxicity, lethality, and distress induction of the substances to an aquatic environment specific to those stages of fish. In many cases, the results (should the chemical end up being harmful) end up as embryo clotting, deformations of the larvae tails, and/or lack of a heartbeat while in the sac.

Currently, there are no test guidelines involving zebrafish as NAMs to evaluate their impact on human health, but this should be reevaluated for a future update or consideration of the list of NAMs available to the public from the EPA. Because the embryos and larvae are transparent, researchers can determine the effects on certain visible organ systems. The heart and surrounding cardiovascular system can be tested within 2-3 dpf for a variety of compounds, and this is a unique advantage to zebrafish embryos compared to rodent embryos because the body of the early fish does not depend on a developed blood circulation system until 5 dpf. The cardiotoxicity and varying phenotypes of the heart already make it a viable NAM that ought to be evaluated by OECD and ICCVAM. Other forms of testing, such as neurotoxicity, hepatotoxicity, and nephrotoxicity are possible from zebrafish that are 6 dpf, younger than their official classification as a vertebrate.

Overall, the potential for zebrafish in its early stages of development for toxicology is substantial and is a growing area of research. The scientific community should consider zebrafish as legitimate alternatives to toxicity testing in mammals and explore how early larval stage zebrafish can be utilized to replace vertebrates.

Works Cited

https://pmc.ncbi.nlm.nih.gov/articles/PMC8707050/

https://www.epa.gov/sites/default/files/2021-02/documents/nams_list_second_update_2-4-21_final.pdf

https://www.oecd-ilibrary.org/environment/test-no-212-fish-short-term-toxicity-test-on-embryo-and-sac-fry-stages_9789264070141-en

https://www.oecd-ilibrary.org/environment/test-no-236-fish-embryo-acute-toxicity-fet-test_9789264203709-en

https://www.epa.gov/assessing-and-managing-chemicals-under-tsca/alternative-test-methods-and-strategies-reduce

https://www.nal.usda.gov/animal-health-and-welfare/animal-use-alternatives#:~:text=The%20%E2%80%9C3Rs%20alternatives%E2%80%9D%20refers%20to,Principles%20of%20Humane%20Experimental%20Technique%22.

https://olaw.nih.gov/sites/default/files/zeb_2016_1344.pdf

https://grants.nih.gov/grants/olaw/guide-for-the-care-and-use-of-laboratory-animals.pdf

https://olaw.nih.gov/sites/default/files/150312_Zebrafish_slides.pdf

https://www.research.uky.edu/division-laboratory-animal-resources/usda-pain-distress-categories

The views expressed do not necessarily reflect the official policy or position of Johns Hopkins University or Johns Hopkins Bloomberg School of Public Health.

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