A venomous fish has a molecule with potential activity against multiple sclerosis

Researchers who developed a toadfish venom antiserum have discovered that the venom contains a peptide that has anti-inflammatory activity and may be used to treat multiple sclerosis.

By Peter Moon  |  Agência FAPESP – Thinking of a poisonous fish usually brings to mind a fugu or blowfish puffed up like a balloon. In such species, which belong to the Tetraodontidae family, the poison is contained within the flesh of the fish. Eating these fish can be fatal unless they have been treated to remove tetrodotoxin.

The blowfish is poisonous but not venomous: it has neither fangs nor spines to inject toxin into victims and paralyze them. The venomous toadfish Thalassophryne nattereri, called niquim in Brazil, has both.

T. nattereri lives in the transition zone between salt and fresh water, hidden in the muddy bottoms of rivers and coastal lagoons. At low tide, this sand-colored fish buries itself in the silt. Out of water, it can survive for up to 18 hours. Anyone who walks along a deserted beach in the North of Brazil or the Northeast region as far south as the coast of Espírito Santo State can be inadvertently bitten by one of these toadfish. Each year, there are reports of 50-100 such accidents along the coast of Brazil. The real number must be greater, because reporting is not mandatory.

In 2008, a group of researchers at the Butantan Institute’s Special Toxinology Laboratory in São Paulo developed an effective toadfish-venom antiserum. Now the same team, led by immunopharmacologists Mônica Lopes-Ferreira and Carla Lima, have discovered that female toadfish, which are smaller than males, nevertheless produce more potent toxin.

The scientists conducted their research at the Center for Research on Toxins, Immune Response and Cell Signaling (CeTICS), one of the Research, Innovation and Dissemination Centers (RIDCs) funded by FAPESP, and have published the results in the journal Toxicon.

In another study, the same group of researchers has found that T. nattereri’s venom contains a peptide (TNP) with activity against multiple sclerosis, a chronic autoimmune inflammatory disorder of the central nervous system in which the immune system attacks the myelin sheath that protects nerve fibers, disrupting communication between the brain and the rest of the organism.

“We demonstrated TNP’s anti-inflammatory action in cases of multiple sclerosis,” Lima said. “In mice, the peptide blocks the passage and infiltration of pathogenic lymphocytes and macrophages into the central nervous system, favoring an increase in regulatory cells. This attenuates neuroinflammation and prevents demyelination, delaying the appearance of symptoms and improving the clinical signs of the disease.”

TNP was discovered in 2007, when Lopes-Ferreira set out to investigate whether T. nattereri’s venom contained peptides as well as proteins. Meanwhile, Lima had standardized laboratory tests in mice for the evaluation of multiple sclerosis. The researchers decided to work together to test TNP’s efficacy in treatment of the disease.

“First, we discovered the peptide’s anti-inflammatory action. More recently, we found that it also acts as an immunomodulator,” Lopes-Ferreira said. Trials to prove the efficacy of TNP for the treatment of multiple sclerosis were conducted at the Butantan Institute’s toxinology lab, in partnership with Cristália, a pharmaceutical company based in Itapira, São Paulo State.

The next steps toward a medical drug require continuation of the partnership with the pharmaceutical company or some other company interested in the discovery and its application, but the researchers are still waiting for their patent application to be processed by INPI, Brazil’s patent office.

“We applied for a patent in 2007, and it hasn’t been granted yet,” Lima said. “Meanwhile, we’ve been granted patents in the European Union, United States, Canada, Mexico, Japan, South Korea, India and China. In all these cases, it took about a year from application to grant on average.”

Females are more venomous

T. nattereri has four spines connected to a gland that produces a potent toxin. In most accidents involving humans, the sole of the foot or the palm of the hand is penetrated by one or more of these spines. The venom causes pain and edema, as well as necrosis, which takes a long time to heal and may lead to loss of function. 

“There are reports of people who weep from the pain. If the foot is pierced by a spine, the swelling makes it almost double in size. The pain and edema can take as long as two months to disappear,” Lima said. These symptoms are caused mainly by natterin, a protease found in the venom.

According to Lopes-Ferreira, natterin initially prevents cellular recruitment. Any inflammatory process recruits and activates phagocytic cells in an initial attempt to control the agent that is causing the problem. Natterin hinders this natural response of the organism. 

Natterin also causes venostasis, or slow blood flow in the veins, a risk factor for forming blood clots, and acts on the extracellular matrix, thus affecting metabolism and interactions among cells.

Female toadfish are now known to be more venomous than males. Males average 22 cm in length and 200 g in weight, whereas females are much smaller, averaging 18 cm in length and 120 g in weight. 

The concentration of toxin in the females’ venom is different and far more necrotizing. As a result, the symptoms of the females’ sting are more severe and lasting. It is not deadly, however. “The venom gland doesn’t produce enough to kill a human being,” Lima said. “It would have to produce 20 times more to do that.”

No pharmacological treatments are available for public use against toadfish venom. Fish venom has a completely different composition from snake or scorpion venom. “T. nattereri’s venom doesn’t belong to the classic toxin family, and the pain it causes can’t be treated with any of the usual painkillers,” Lima said. 

Because one of Butantan Institute’s specialties is producing snake antivenom, in 2008, the Toxinology Lab team inoculated horses with toadfish venom and used the equine antibodies to generate an antiserum. This antiserum proved to be effective in mice, neutralizing necrosis and pain and reducing edema.

“The fact that it inhibits necrosis is important enough,” Lopes-Ferreira said. “Necrosis is one of the most disturbing effects of an accident with this venomous species.” The antiserum is not yet being produced because industrial-scale production requires capex financing from the Ministry of Health and the São Paulo State Department of Health. 

The article “Analysis of intersexual variation in Thalassophryne maculosa fish venoms” (doi: http://dx.doi.org/10.1016/j.toxicon.2016.02.022) by Mônica Lopes-Ferreira, Ines Sosa-Rosales, Fernanda M. Bruni, Anderson D. Ramos, Fernanda Calheta Vieira Portaro, Katia Conceição and Carla Lima can be retrieved from sciencedirect.com/science/article/pii/S0041010116300393.