Soft corals palitoxin: Mouse bioassay and hemolytic assay.

Palitoxin (PTX) is a potent water-soluble toxin produced by some soft corals, so named because, unlike hard corals, they do not generate a skeleton of calcium carbonate, so they are not reef generators. The producing species belong mainly to the genera Palythoa and Zoanthus. Palitoxin can also be produced by some species of algae, dinoflagellates (genus Ostreopsis) and other microorganisms.

The toxin was isolated, named and first described by Moore and Scheuer in 1971 from the coral species Palythoa toxica. It is a polyhydroxy compound and partially unsaturated with a long carbon chain, with 64 chiral centers. Due to the chirality and the possible double-bond cis-trans isomerism, it has more than 1,021 alternative stereoisomers. It is thermostable and the treatment with boiling water does not eliminate its toxicity. It remains stable in aqueous solutions for prolonged periods, but it decomposes rapidly and loses its toxicity in acidic or alkaline solutions. It has multiple analogues with a similar structure, such as ostreocin-D or mascarenotoxin-A and -B.

The toxin producing organisms are found in the tropics and subtropics. In addition, it can also be found in many other animal species, such as fish and crabs, due to the biomagnification process, whereby it is concentrated in the tissues of tolerant organisms at successively higher levels in a food chain. It can also be found in other organisms that live near palitoxin-producing species, such as sponges, mussels, starfish and cnidaria.

Palitoxin poisoning is relatively rare, and most cases occur from eating fish or shellfish that has accumulated sufficient levels of that toxin. Cases have also been documented in people exposed to proliferation of certain algae. On the other hand, aquarium related poisonings occur as a result of skin exposure after prolonged handling of corals, through small wounds or abrasions on the hands, or inhalation of aerosols while cleaning the aquarium or coral is manipulated. Corals of the genus Palythoa are often recommended to new owners of marine aquariums because they are considered relatively easy to maintain. Fans and aquarium providers sometimes do not know or underestimate the health risks associated with maintaining soft corals in home aquariums because there are few documented cases of poisoning.

Although the exact mechanism of action is unknown, it is postulated that palitoxin binds to the Na⁺/K⁺ ATPase pump, a transmembrane protein found on the surface of all vertebrate cells, transforming it into an ionic channel through the which Na⁺ and K⁺ ions can flow easily in favor of their concentration gradient (approximately one million times faster than the pump itself can move them in the opposite direction). The concentration of sodium in the cytoplasmic space increases to equalize the extracellular concentration while that of potassium decreases in the cytosol to equalize the extracellular level, which finally paralyzes the active movement of ions. Usually, when cells gain sodium ions, they also gain chloride ions and this net salt gain generates an osmotic flow of water into the cells that ultimately results in cell lysis. The sodium-potassium pump is necessary for the viability of all cells, and this explains the fact that palitoxin can affect multiple organs and cause serious symptoms, including death.

It is difficult to know if a coral of this type produces the toxin, therefore, if you have them, what should be done is to assume that this is the case, and take the following measures in this regard:

• Handle corals as little as possible.

• Do not touch the coral with bare hands. Wear goggles and protective gloves whenever corals are handled. Latex gloves break easily when sharp rocks are handled, so long thick rubber gloves that protect the hands and forearms are recommended. 

• Always handle corals underwater. If you have to take it out of the aquarium, put it in a bag in the water, and if possible in low light (the effect of heating in shallow water can trigger the release of palitoxin), while using protective equipment and sealing the bag before taking it out of the aquarium.

• Ensure that the aquarium water level is sufficient so that the coral surface is completely submerged at all times.

• Avoid any coral maintenance activity that may release aerosols or sprays, such as breaking, cutting, scrubbing, scraping, brushing and using boiling water or chemicals. 

• Avoid placing corals under strong lights, especially when out of the water. They can cause heat stress, causing rapid release of palitoxin.

• Avoid splashing and contaminating nearby surfaces when changing tank water, and dispose of aquarium water carefully.

At present there is no recognized official method for the determination of palitoxin, so that each laboratory, according to its possibilities, develops its particular methodology, making it necessary to combine several methods, as the physical-chemical or biological tests, in order to confirm the presence of palitoxin in a sample. Biological assays are generally more sensitive, and among them are:

• Bioassay in mice: the crude extract that supposedly contains the toxin is injected intraperitoneally into the mouse. The animals are kept under observation and the times of onset of symptoms, and where appropriate, of death, are noted. This type of test allows to characterize the toxins present in a sample, but does not allow its quantification. 

• Hemolytic assay: it is a fast and sensitive method to determine palitoxin. This toxin has the ability to slowly lyse red blood cells, releasing hemoglobin into the medium, the concentration of which can be determined by measuring absorbance at 405 nm. This technique makes it possible to quantify palitoxin levels approximately. Confirmation of the toxin can be done either using a specific antibody or an antagonist (ouabain). 

Tests performed in IVAMI

• Bioassay in mice.
• Hemolytic assay.