International Journal of Drug Development and Research

Keywords:

Scorpaenidae, Venom, Cardiovascular, Neuromuscular, Hemolyti

INTRODUCTION

Venomous and poisonous animals are also found in aquatic environment in large number. Among these, venomous fish attracts special interest, since they represent more than 50% of venomous vertebrates and are often involved in human accidents 1, 2, 3. Russell, in 19964 reported around 200 species of marine fish, including stingrays, scorpionfish, zebrafish, stonefish, weeverfish, toadfish, stargrazers and some species of shark, ratfish, catfish, sugeonfish and blenny which are known or suspected to be venomous. But this number of fish species clearly underestimates the number of venomous fishes implied by the phylogenetic distribution of venom among ray-finned fishes3. The most dangerous,

venomous fish known belong to Scorpaenidae family and, according to the venom organ structure, they are divided in to three groups typified by different genera: Pterois (lionfish), Synanceja (stonefish) and Scorpaena (scorpionfish)5.

Marine fish belonging to family Scorpaenidae cause severe injuries and sometimes death in humans around the world6. Studies on the epidemiological and clinical aspects of envenomation caused by these fishes focused mainly on lionfishes and stonefishes 7. However, there are few reports of injuries caused by the venom ejected by the venom apparatus of Scorpaena 8.

Three populations are at higher risk for spiny fish envenomation: fishermen sorting the catch from nets, waders, and aquarists etc. Lionfish (genus Pterois) are common to home aquariums and responsible for envenomation when mishandled. At least eighty species of the family scorpaenidae are said to possess venom apparatus. Fishermen and swimmers are the main victims of scorpionfish envenomations which occur as a result of contact with sharp dorsal pelvic or anal spines and stonefish envenomation is still an occupational hazard in many areas of the world 9. The distribution and number of venomous fish belonging to Scorpaenidae family has been listed in Table 1.

Source: Adapted from Khora, 198654; Smith and Wheeler, 20063

SCORPAENIDAE VENOM APPARATUS AND ENVENOMATION

All the fishes of family Scorpaenidae have 11- 17 rays or spines in the dorsal fin (in the genus Scorpaena, there are 12-13 rays) 10. The dorsal spines of Lionfishes are slender and long and contain small venom glands. Whereas, the spines of scorpionfish are strong and short, and the venom glands are more developed. The spines of stonefish are very thick and short with highly developed venom glands 11. The venom apparatus of the Scorpaenidae is constituted by elongated venom glands in the anterolateral grooves of the spines in the dorsal, pelvic and anal fins without an excretory duct in the case of lionfish and scorpionfish while in the case of stonefish, there are highly developed longitudinal paired glands with well-developed duct like extensions 8. It is thought that venom apparatus evolved relatively recently in the development of venomous fish, because while fish are on a higher level of development than some other groups of venomous creatures (e.g. spiders), their venom apparatus are much more primitive relying on a completely involuntary mechanical action, rather than a voluntary expulsion of venom 12.

Venom is delivered when spines pierce the tissue of the victim, glands get compressed, the integumentary sheath enclosing the spine is ruptured and the venom enters the wound 13. The envenomation caused by these fishes reported various symptoms including intense pain, swelling, ulcers, skin damage and rarely death associated with bacterial infection 14. It may take a few days or several weeks for recovery from the local effects (Table 2). Hot water immersion or heat therapy for few minutes can be provided as firstaid.

SCORPAENIDAE VENOM

Like terrestrial venomous animals, fish venoms also contain a variety of pharmacologically active component. A very less research has been done on venomous fishes, though the symptoms produced by their envenomation are severe. It may be due to less threat associated with marine creatures in respect to terrestrial animals and also due to extreme instability and scarcity of the venom 19. The venom produced by these fishes is diverse and chemically complex in nature. Fishes of the family Scorpaenidae contain heat labile venom which is common in all species but with different potency and possess cardiotoxicity and neurotoxicity. The proteinaceous nature of venom is still not well characterized. There are few studies on the venom of the scorpionfishes (Scorpaena). The venom is highly heat labile, the lethal property being associated with the proteinaceous fraction of venom having molecular weight more than 50,000 and less than 800,000. This separated fraction is more lethal than crude venom 20. It is reported that the fluid ejected from the blister caused by Lionfish sting produced experimental platelet aggregation 21. Three main toxins have been isolated from various species of stonefish: stonustoxin (SNTX), verrucotoxin (VTX), and trachynilysin (TLY). Stonustoxin (SNTX), from S. horrida, has 2 subunits, α and β (71,000 and 79,000 Daltons, respectively) 22 (Table 3). It induces formation of hydrophilic pores in cell membranes. Toxicity in animals includes hemolysis, local edema, vascular permeability, platelet aggregation, endotheliumdependent vasodilation, and hypotension. It is described that the pronounced effects of stonefishes venom are on the cardiovascular and neuromuscular systems, with hemolytic and hyaluronidase activities 23. Stonustoxin (SNTX) which is toxic protein with lethal action has been identified which causes severe hypotension. It is also found possessing high cholinesterase, acid and alkaline phosphatase and phosphodiesterase activity 7.

EFFECTS ON CARDIOVASCULAR SYSTEM

Piscine venoms have potent activity. It is observed that the venom of the Indian stonefish (S. horrida) produces marked cardiovascular changes in experimental animals 30 and also found that the cardiovascular pharmacology of S. trachynis venom have produced results that have differed from those obtained by researchers using the venoms of S. horrida and S. verrucosa 31. It is found that lyophilized venom produced endothelium-dependent relaxation in vascular smooth muscle of the rat 28. The same effect has also been observed in the lethal fraction of S. horrida venom i.e., stonustoxin32. They also found that stonustoxin (SNTX) at low concentration responsible for causing endothelium dependent relaxation and at high concentration; it shows endothelium independent contraction33. Both vascular and non-vascular smooth muscles are affected by many fish venoms.

Clinical and epidemiological studies on the S. plumieri venom shows pronounced cardiovascular effects8. The scorpionfish (S. plumieri) at sub-lethal doses (14-216 μg/Kg) in the anesthetized rat produces hypertensive response. While at higher lethal doses (388 μg/Kg), this response is followed by hypotension, a visible respiratory difficulty which ends up in respiratory arrest34. It is concluded that the respiratory collapse is actually, a consequence of cardiovascular collapse. The effect of piscine venoms on isolated vessels is found to be concentration dependent. It is found that S. trachynis venom produces a relaxation at low concentration and at high concentration produces contractile effect28. The venom of the lionfish P. volitans produces a marked hypotensive effect in anesthetized rabbits35.

Church and Hodgson in 2001 worked on P. volitans venom to determine its cardiovascular effects as well as to elucidate the mechanism of action of venom. Venom produces dose and endothelium-dependent relaxation in pig coronary artery 36. Cardiovascular activity of Scorpaena plumieri venom was investigated by both in vivo and in vitro model 34. The results of their experiment shows that, in the anesthetized rat, the scorpionfish venom produces a remarkable hypertensive response at sub-lethal doses (14-216 mg/kg), follows by hypotension at higher or lethal doses (338 mg/kg), respiratory difficulties are also visible which ends up in respiratory arrest. Stonefish envenomation symptoms are similar to soldierfish envenomation symptoms which include severe burning pain, local swelling, sweating, nausea and loss of perceptiom37. As the envenomation symptoms of soldierfish and stonefish share similar pharmacological activity and qualitative similarity, so the effectiveness of antivenom (raised against the venom of stonefish; Synanceja spp.) in neutralising the cardiovascular effects of soldierfish (G. marmoratus ) is investigated2 and it is found that the Synanceja antivenom is effective in neutralising most of the cardiovascular activity of G. marmoratus venom extract in vitro and in vivo both. This suggests that the Synanceja antivenom may have potential which can be used in severe cases of envenomation caused by G. marmoratus.

EFFECTS ON NEUROMUSCULAR SYSTEM

Though the symptoms of Synanceja envenomation are mostly cardiovascular in nature, some symptoms related with neuromuscular effects have also been reported38. Electrophysiological and electron microscopic examination of isolated murine and frog nerve-skeletal muscle preparations exposed to various concentration of stonefish venom shows the neuromuscular toxicity of the venom. It is observed that the low concentrations of venom acts presynaptically (Na+ channel-independent) while higher concentrations of venom acts presynaptically and postsynaptically both by causing release and depletion of neurotransmitter from the nerve terminal39. This neurotransmitter release is found to be resistant to blockade of Na+ channel and also resistant to botulinum toxin. This indicates the release of acetylcholine via a non-exocytotic mechanism. The crude S. trachynis venom is found to increase the mean number of quanta released per nerve stimulus and produces markable damage to nerve and muscle cells, as well as preventing synaptic recycling at higher concentrations in the frog stimulated cutaneous pectoris nerve-muscle preparations. From these observations, it is concluded that the crude venom of S. trachynis is responsible for neurotoxic effect by releasing massive neurotransmitter at low concentrations and causes muscle and nerve damage at higher concentrations 39. In the same study it is found that the lethal fraction, TLY of S. trachynis crude venom is responsible for neuromuscular effects in vitro. Also, TLY has been shown to induce Ca+2 entries in to adrenal chromaffin cells and also affecting intracellular Ca+2 stores and increasing Ca+2 dependent catecholamine releases40. Signs of neurotoxicity have also been observed both in mice and other fish by Pterois volitans venom24. It is observed that P. volitans venom extract derived from its spine tissue decreases the heart rate and force of contraction in isolated clam and frog hearts. These actions are found to be due to presence of micromolar concentrations of acetylcholine in the extract. The hydrolysis of acetylcholine by exogenous acetylcholinesterase does not affect the venom toxicity as well as the heart function. This indicates that the venom is interfering with neuromuscular transmission rather than the muscle itself. Unaffected sodium channel blockade suggests that the toxin has an effect at the nerve terminal rather than interfering with synaptic transmission41.

The direct evidence of trachynilysin, isolated from S. trachynis venom is observed, which increases the spontaneous quantal acetylcholine release in Torpedo nerve-muscle preparations42. It is found that the lyophilised venom produces endothelium-dependent relaxation in the vascular smooth muscle of the rat. The same effect has also been observed in studies using stonustoxin (SNTX), the lethal fraction of S. horrida venom32. The venom of Scorpaena guttata causes a biphasic response in paired atria consisting of an inotropic decrease due to activation of muscarinic receptors and follows by inotropic increase due to activation of adrenoceptors 43. It is known that the cardiovascular effects associated with stonefish envenomation are life-threatening37, however it is not known whether the lethality of stonefish venom is due to the cardiovascular effects, or to neuromuscular toxicity. Studies conducted on S. verrucosa crude venom shows neurotoxic effect when the venom is administered i.c.v. to rats. This effect is found to be different from the VTX, the lethal toxin isolated from S. verrucosa venom. This fraction is unable to produce the same severe neurotoxic effect as produced by crude venom when administered i.c.v. to rats suggesting the presence of at least one other active component in S. verrucosa crude venom44. SNTX similar to TLY produces a contracture followed by a decrease in electrically-evoked twitches in the mouse hemi-diaphragm and chick biventer cervicis muscle as well. It is concluded that the contraction produced by SNTX is not dependent on the release of acetylcholine but rather Ca+2 release and / or activation 33.

G. marmoratus envenomation in mice leads to neurotoxic effects which include paralysis of hind limbs, muscular weakness and at higher doses lead to coma and respiratory cessation. Also, G. marmoratus venom reduces the response of skeletal muscle to nerve stimulation but the mechanism involved in this is not studied 45. Studies conducted by Church et al., 2003 46 on the venomus fishes of the family scorpaenidae show for the first time that the crude venoms of G. marmoratus and P.volitans produce contraction in the chick biventer cervicis muscle. They also find that all three crude venoms produce an increase in intracellular Ca+2 in murine cortical neurons. The increase in Ca+2 is thought to be mediated via the formation of nonselective cationic pores in the cell membrane and is dependent on the entry of extracellular Ca+2.

HEMOLYTIC ACTIVITIES

Cytolytic toxins are well known for their ability to kill cells. Fish venoms have hemolytic activity. The stonefish (Synanceja verrucosa) crude venom is found to be highly hemolytic to rabbit erythrocytes but the activity is reduced with time even when the venom is frozen. The crude venom is also purified and verrucotoxin is isolated by DEAE and hydroxyapatite chromatography. Verrucotoxin is found to be lethal and cytolyitc 47. Neoverrucotoxin (neoVTX) is a proteinaceous toxin obtained from the venom fluid of S. verrucosa, has hemolytic and lethal activities and is inhibited by anionic lipids 48. The venom of other stonefish Synanceia trchynis is also studied. The venom contains a cytolytic toxin which is antigenic and precipitated by ammonium sulphate. The toxin is found to be potent but narrow-spectrum in action which is lytic for erythrocytes of rabbit, dog, rat and guinea pigs in vitro but it is totally or largely inert against the erythrocytes of sheep, cow, human, monkey, mouse, goat, horse, burro and cat (Table 3). The hemolytic activity of venom does not get separated from its lethal and vascular permeability-increasing activities by fractionation of venom using molecular sieve fast protein liquid chromatography and isoelectric focusing. These activities of venom are found to be sensitive against heat and protease treatment50. P. volitans venom has been found to possess hemolytic activity and is highly selective to rabbit erythrocytes 51. Stonustoxin (SNTX), purified from the crude venom of stonefish (Synanceja horrida) has potent hemolytic activity. The pore forming property of stonustoxin was examined by osmotic protection assay. It is found that the SNTX causes the lysis of erythrocytes by the formation of hydrophilic pores in the cell membrane. In the case of SNTX, positively charged lysine and arginine residues are used where the modification of positively charged side chain shows the inhibition of hemolytic activity of SNTX. Also the hemolytic activity of SNTX is competitively inhibited by various negatively charged lipids51. It is demonstrated that the reduction in number of free tryptophan residues is responsible for impairing the hemolytic activity of SNTX and it can be correlated with other toxins that require tryptophan residues for cytolytic activity52. The proteolytic enzymes are found to be present in the venom of S. plumieri responsible for hydrolyzing other bioactive proteins 53.

CONCLUSION

Many cases of scorpaenidae envenomation have been reported in different parts of the world. Fishes of the family scorpaenidae are responsible for severe injuries but their venom contains active components which are of pharmacological importance. Scorpaenidae venoms have been recognized as potential source of pharmacological agents and physiological tools. Their venom interacts with physiologically important molecular targets and affects the vital function of organisms. In most cases, toxin present in venom is responsible for physiological effects. Scorpaenidae venom produces distinct cardiovascular changes. Stonustoxin is responsible for causing endothelium dependent relaxation at low concentration and shows endothelium independent contraction at high concentration. P. volitans venom produces hypotensive response whereas S. trachynis venom produces hypertensive response. The lethal toxin of S. trachynis venom (TLY) is responsible for the release of acetylcholine from the neuromuscular junction. Some of the toxins have neurotoxic effects which includes paralysis of hind limbs, muscular weakness and at higher doses causes coma and respiratory failure. Many of the fish venoms contain toxin responsible for erythrocyte lysis. The mechanism behind erythrocyte lysis by SNTX is through the formation of hydrophilic pores in the cell membrane. P. volitans hemolytic activity is found to be selective to rabbit erythrocytes. SNTX activity gets altered as the numbers of tryptophan residues get decreased. Scorpaenidae fish venom possess different properties which can be utilized for research tools and potential drug development.

Tables at a glance

Table 1 Table 2 Table 3 Table 4

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