Every day, scientists make discoveries that help improve human health and wellness. Many medicines are discovered in nature, like toxins from animals. We decided to study a fish called Copper Joe toadfish (Thalassophryne nattereri) and found that a small molecule in the fish’s venom, called TnP, can help damaged organs and tissues. However, before any molecule can be used in humans, it must be tested on laboratory animals. These tests are called pre-clinical trials and are important to prove that a treatment is safe for humans. Our study tested TnP in zebrafish and found that it is safe and has no toxic effects on the developing fish. In the future, scientists can continue researching TnP, and hopefully, someday, it will be developed into a medicine to help thousands of people around the world who are suffering from diseases that cause inflammation.
Some Fish Are Poisonous
Did you know that there are many kinds of poisonous fish? Would you believe that the venom of one of these fish contains a substance that can treat diseases that cause inflammation? It is true! The venom present in poisonous fish is a mixture of many molecules. The fish use these important molecules to defend themselves from their enemies (other animals that want to feed on them) and to protect the areas where they live, by scaring away invaders to defend their territory.
Thalassophryne nattereri is the scientific name of a poisonous fish in Brazil, which causes many incidents involving fishers and sunbathers. It is mainly found in the north and northeast regions of Brazil and is popularly known as niquim, or Copper-Joe toadfish. The niquim is a small fish, measuring about 15 cm in length; it likes to spend most of its time buried in the sand in brackish (slightly salty) water, where rivers meet the sea. This venomous fish has four spines, one on each side of its body, and two on the top. These spines are naturally hollow, like straws, and when the spines are pressed, poison from venom glands comes out of them (Figure 1). Incidents can happen when people are walking along the beach, swimming in the sea, or even when the fish get caught on fishing hooks or in nets. If a person steps on a niquim or accidently touches the spines, the poison comes out of the fish and goes into the human. These incidents cause extreme pain, and the region where the poison enters becomes swollen and red. Within a week the tissue around the wound starts to die, becoming necrotic .
Our group has been studying the niquim and its venom since 1996, and we have made many important discoveries. We were the first to identify the main molecules present in the venom. We named them nattectin and natterins. Natterins are a set of proteins (there are five of them), but the venom only has one nattectin protein. Together, these molecules are responsible for the pain, swelling, and necrotic tissue that develop in humans that have been in contact with the fish’s venom. Interestingly, we recently discovered that natterins are present in many other organisms, and their function is to protect those organisms .
A Great Discovery!
We discovered another important small molecule in the venom of the niquim—a peptide that we named TnP, for Thalassophryne nattereri peptide. Unlike natterins and nattectin, we found that TnP was not involved in the symptoms observed in people that have niquim incidents. In fact, our studies showed that TnP inhibited inflammation. Let us remember that inflammation is a response of the body to harmful stimuli, diseases, or infections by bacteria or viruses. The finding that TnP inhibited inflammation was a very interesting discovery, so we did additional studies to find out more about TnP.
First, we examined TnP’s effect in animals. We started with mice, and those experiments helped us discover that TnP decreases inflammation caused by two critical diseases that many people worldwide suffer from: asthma and multiple sclerosis. Asthma is a disease of the respiratory system, and multiple sclerosis is a disease that affects the central nervous system. TnP proved effective in protecting against the development of these diseases, relieving their symptoms, and did not cause side effects.
From the Lab to a Medicine
Our results increased our confidence that TnP could be used as a medicine to treat inflammatory processes, but first it was necessary to investigate its safety—for a substance to become a medicine, it must be proven to be safe for humans. This safety testing, called a pre-clinical study, is also performed in animals. The pre-clinical study is an essential step in developing a new drug (Figure 2).
For our pre-clinical study, we used zebrafish, a little fish native to South Asia and that shares 70% of its genes with humans and is currently widely used as an experimental laboratory animal for drug development . Zebrafish have numerous advantages, including rapid growth, a transparent embryo-larval stage, and a small size .
TnP Is Safe and Effective
To ensure the safety of TnP, we first exposed groups of zebrafish embryos to various doses of TnP. We monitored the embroys/larvae every day for 4 days to look for any defects in development. For comparison, we examined a separate group of fish embryos that were not exposed to TnP (Figure 3). Then, we checked whether the hearts of the zebrafish larvae were beating correctly, whether the larvae were swimming normally, and whether the larvae’ brains were well-developed . All these tests confirmed that TnP did not cause heart or brain damage in the embryos that were exposed to it.
Our studies allowed us to conclude that TnP, a molecule from the venom of the fish niquim, is safe and effective and could be a promising drug for the treatment of human diseases that involve inflammation, such as asthma and multiple sclerosis. The next step is continuing testing TnP to better understand how does it helps in the immune response, and clarify its mode of action. In the future, we will continue to work with TnP, evaluating its ability to inhibit the inflammatory process of other important human diseases.
Inflammation: ↑ A response of the body to harmful stimuli, diseases, or infections by bacteria or viruses. Redness, heat, swelling, and pain are common symptoms during inflammation.
Necrotic: ↑ When most or all of the cells in an organ or tissue die due to disease, injury, or failure of the blood supply.
Nattectin: ↑ A kind of lectin protein isolated in the niquim venom that binds to sugar molecules (carbohydrates) and is involved in inflammation.
Natterin: ↑ New proteins discovered in the venom of the fish Thalassophryne nattereri, which are the most abundant proteins in the venom and the main responsible for the envenomation symptoms and wounds in humans.
Peptid: ↑ Short chains of amino acids, the same parts that together form the proteins that play critical roles in the biological functions of all kinds of organisms.
TnP: ↑ Thalassophryne nattereri peptide, a peptide discovered in the venom of the niquim that helps to treat inflammation.
Pre-clinical Study: ↑ Research using laboratory animals to determine if a drug, procedure, or treatment is effective and safe before it is tested on human volunteers.
Embryo-Larval Stage: ↑ The earliest developmental stages of fish. The embryo stage includes development in the egg, while the larva stage occurs right after the fish leaves the egg and starts to swim.
Conflict of Interest
The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.
We thank the São Paulo Research Foundation (FAPESP) for the support, notably through the Center of Toxins, Immune Response, and Cell Signaling—CeTICS (grants #2013/07467-1; #2019/27677-7) and its dissemination branch. Also, huge thanks to the Zebrafish Platform staff and collaborators. Special thanks to the young reviewers and mentors for the meaningful feedback.
Original Source Article
↑Batista-Filho, J., Falcão, M. A. P., Maleski, A. L. A., Soares, A. B. S., Balan-Lima, L., Disner, G. R., et al. 2021. Early preclinical screening using zebrafish (Danio rerio) reveals the safety of the candidate anti-inflammatory therapeutic agent TnP. Toxicol. Rep. 8:13–22. doi: 10.1016/j.toxrep.2020.12.004
 ↑ Lopes-Ferreira, M., Grund, L. Z., and Lima, C. 2014. Thalassophryne nattereri fish venom: from the envenoming to the understanding of the immune system. J. Venom. Anim. Toxins Incl. Trop. Dis. 20:35. doi: 10.1186/1678-9199-20-35
 ↑ Lima, C., Disner, G. R., Falcão, M. A. P., Seni-Silva, A. C., Maleski, A. L. A., Souza, M. M., et al. 2021. The Natterin protein family diversity: a review on phylogeny, structure, and immune function. Toxins. 13:538. doi: 10.3390/toxins13080538
 ↑ Howe, K., Clark, M. D., Torroja, C. F., Torrance, J., Berthelot, C., Muffato, M., et al. 2013. The zebrafish reference genome sequence and its relationship to the human genome. Nature. 496:498–503. doi: 10.1038/nature12111
 ↑ Novoa, B., and Figueras, A. 2012. Zebrafish: model for the study of inflammation and the innate immune response to infectious diseases. Adv. Exp. Med. Biol. 946:253–75. doi: 10.1007/978-1-4614-0106-3_15
 ↑ Batista-Filho, J., Falcão, M. A. P., Maleski, A. L. A., Soares, A. B. S., Balan-Lima, L., Disner, G. R., et al. 2021. Early preclinical screening using zebrafish (Danio rerio) reveals the safety of the candidate anti-inflammatory therapeutic agent TnP. Toxicol. Rep. 8:13–22. doi: 10.1016/j.toxrep.2020.12.004