SNAKE VENOMS

SNAKE VENOMS
Snake venoms contain complex mixtures of various organic materials (proteins, peptides, amino acids, and other compounds). Snake venoms have been of interest to scientists and laypersons alike for thousands of years, but only during the last 3 decades has biochemical technology allowed isolation and precise identification of the toxins (venins, constituents) in venoms. Isolation of specific toxins has enabled scientists to study the role of each toxin in the overall action of the venom.

A basic understanding of venoms is necessary to understand the rationale for certain treatment regimens. Hundreds of research papers and monographs have been written on the subject of snake venoms by protein biochemists, toxinologists, toxicologists, physiologists, pharmacologists, immunologists, and medical scientists. This short discussion is meant only to provide an introduction and overview of this rapidly expanding area of venom research. More detail may be obtained from reviews of the subject.

Though much of snake venin research has been directed at finding solutions for problems associated with envenomation of humans, most research studies have employed experimental animals or isolated tissue. Different species may react differently to venins; still, much can be learned about envenomation in general by understanding the effects of snake toxins on mice, rabbits, sheep, and other animals.

The primary toxins of snake venoms are proteins and peptides. New toxins are being described almost monthly. Table I lists known toxins according to biochemical components, while Table 2 classifies toxins according to their primary effect on mammalian tissues or organ systems. Toxins are usually named according to the snake venom from which the toxin was first isolated, or the primary pharmacologic effect on mammalian victims.9 Many toxins have not yet been precisely identified and are currently designated only as fraction x, y, or z from a given source. Snake venoms may vary, even between closely related species. Toxin nomenclature is anything but standardized, and multiple names may exist for the same toxin studied in various areas of the world. The author has followed the nomenclature found in Tu's Reptile Venoms and Toxins.

Enzymes are the major toxic components of snake venoms. Phospholipase A2 activity (i.e., hydrolytic breakdown of membrane phospholipids) is basic to the action of many of the toxins in snake venoms. Pharmacologically, phospholipases A2 may have neurotoxic, cytotoxic, and anticoagulant activities. As a presynaptic neurotoxin CP-neurotoxin), phospholipase A2 acts on peripheral axon terminal membranes in such a way as to decrease the release of acetylcholine into the neuromuscular junction. Post-synaptic neurotoxins (a.-neurotoxins) bind to acetylcholine receptors on post-synaptic membranes to prevent uptake of the neural transmitter. Both pre- and post-synaptic neurotoxins effectively paralyze skeletal muscles, including the diaphragm, thereby immobilizing and asphyxiating the victim.

Cytotoxic phospholipases A2 form lysolipids that act like detergents in lysing cells, causing necrosis of muscle tissue (myotoxins). As anticoagulants, phospholipases A2 eliminate the procoagulant phospholipids before blood clotting factors can be activated and begin amplification of the clotting cascade. 13 Descriptions of more detailed biochemical reactions are beyond the scope of this book.

Many P-neurotoxins have been recognized in venoms of elapid snakes, but they are also found in crotalid and viperid . These toxins effectively block nerve impulse transmission at the presynaptic location and paralyze the victim.  Some of the most potent toxins known are a.-neurotoxins, usually found in elapid snake venoms. Although a.-neurotoxins act at the post-synaptic location by binding to nicotinic acetylcholine receptors, the effect is again a paralysis of the peripheral nervous system.

Cardiotoxins may act independently or synergistically with phospholipase A2 , particularly in causing a lytic effect on erythrocytes. Cardiotoxins cause prolonged muscular contracture with subsequent loss of the ability of cardiac muscle to contract in response to normal stimuli. Russell 19 lists the following biological activities of cardiotoxin: blockade of neuromuscular transmission, blockade of axonal conduction, membrane depolarization, anticholinesterase action, local tissue action, hemolysis, cytotoxic action, skeletal muscle contracture, smooth muscle contracture, and cardiac arrest. The biochemical method of action is unknown.

Complement is not a single entity, but a complex proteinaceous enzyme system or cascade facilitating antibody-mediated reactions and induction of major inflammatory pathways. A cobra venom factor (CVF) that had a lytic effect on erythrocytes was identified at the end of the 19th century. Using modem biochemical technology, CVF has been purified and tested for physiologic action. Administered intravenously, CVF has little toxic effect, but its action at a bite site triggers activation of complement, releasing anaphylatoxins which in tum cause an increase in vascular permeability. Increased vascular permeability aids in the absorption of toxins into the bloodstream.

Venom platelet-active principles include proteins, peptides, and glycoproteins that may either stimulate or inhibit platelet action. Agents that stimulate platelets include phospholipases A2, serine proteases, and glycoproteins. Inhibitory toxins also include phospholipase A2 activity and glycoproteins, plus peptides and fibrinogenases. Platelet-active toxins are more common in the venoms of viperids and crotalids than in elapid snake venoms. These actions may contribute to thrombocytopenia and/or hemorrhage.

Thrombin-like venom enzymes induce the clotting of fibrinogen  These enzymes are primarily found in crotalid venoms, less so in viperid venoms. Certain toxins (e.g., Russell's viper venom-V and thrombocytin) activate Factor V in the blood coagulation cascade. Other snake venoms activate protein-C in the coagulation sequence. Certain factors in snake venoms may inhibit or stimulate the clotting mechanism, contributing to disseminated intravascular clotting (DIC) or hemorrhage.

Envenomation by crotalid snakes usually results in a marked local response  characterized by erythema, hemorrhage, edema, and necrosis. Response at the bite site is visible, but identical action may take place in the brain, lung, kidney, heart, or liver, quickly in severely affected victims, or as the effects of envenomation progress. This progressive effect should be kept in mind when  monitoring patients or contemplating the use of antivenin. It is not true that antivenin has no inhibitory effect on local reactions to snakebite. Hemorrhagic toxins attach to endothelial cells of blood vessels causing swelling and rupture of membranes, resulting in hemorrhage. Hemorrhagic toxins are proteolytic in action. Myonecrosis may be the consequence of the action of myotoxins on the sarcoplasma of muscle cells.

Although specific nephrotoxins have not been identified, there is ample evidence that nephrotoxicity occurs in snakebite, particularly from the venom of such viperid snakes as Russell's viper. The cytotoxic effects of crotalid venoms may also cause renal lesions. The range of renal lesions includes acute tubular necrosis, acute cortical necrosis, glomerulonephritis, and acute interstitial nephritis. Renal failure may develop late in the course of an envenomation, another reason for continual monitoring of the patient for signs of parenchymal organ dysfunction. Signs of renal involvement include hemoglobinuria, myoglobinuria, disseminated intravascular coagulation, hypotension, and hemorrhage. Aside from the importance of their poisonous effects, snake venoms have proven to be valuable tools in the study of nerve conduction, blood coagulation, tumor growth, and enzymology.

The primary method of determining the toxicity of snake venoms and individual toxins is to establish the lethal dose in 50% of the experimental animals of a given species into which the venom is injected (LD50). The mouse, Mus musculus, is the usual subject. A perusal of the literature quickly reveals great variation in the values reported by various authors. The method of collection, time of collection, and how the venom is handled following collection may have a significant bearing on the potency of the venom.

Toxicity as established in the laboratory may differ markedly from the effects of an actual bite. Table 4 lists variables that may modify the ultimate effect of a venom. While some data on the toxicity of venoms for laboratory species is available, little factual data has been collected on toxicity for domestic and wild animals. The primary function of snake venom is to procure food. Most venomous snakes stalk prey, strike, and release, waiting for immobilization to occur before grasping and ingesting the animal. Venom also serves a beginning digestive function. Secondarily, venom may be used defensively.

ELAPID SNAKES
The elapids have front-fixed fangs (proteroglyphous). The length of the fangs varies from species to species, with the Sea snake and North American coral snakes having short fangs. Fangs are partially covered by a membrane that is pushed away at the time of the bite. Elapid fangs are either grooved or have a closed groove that functions in the same manner as a hollow fang. The venom duct empties at the base and bathes the fang. Although there is species variation, the venom gland (homologous with the mammalian parotid salivary gland) generally lies ventral to the eye, dorsal to the upper lip, and extends caudally to the commissure of the mouth. Usually, a single duct connects the gland to the base of the fang. Smaller elapid snakes bite and continue to chew while holding on to the victim. Larger snakes may bite repeatedly, especially in a defensive situation. When agitated, cobras rear up to face danger. Striking distance is measured by the height of the head above the ground because the snake strikes forward and downward, with the fulcrum being the point of contact of the snake with the ground. Cobras are relatively slow in striking speed, which enables the mongoose, Herpestes spp., family Viverridae, to dodge and ultimately grasp the snake. A few elapid snakes (e.g., spitting cobras) have evolved a novel means of defense, utilizing venom and specialized fangs that have a distal orifice directed at a right angle to the fang. When threatened, the snake elevates the head  and sprays two jets of venom toward the eyes of the animal. The venom may be projected accurately a distance of 3 to 4 m. The venom causes an immediate conjunctivitis and keratitis that results in temporary blindness. Spitting cobras immobilize prey by biting in a normal cobra manner.

VIPERID AND CROTALID SNAKES
Viperid and crotalid snakes have elongated, hollow, front fangs (solenoglyphous) that lie folded on the roof of the mouth, covered by a membranous sheath, when not in use (Figures 2 and 3). Location of the venom gland is similar to that of elapid snakes. 30 A primary, convoluted duct connects the venom gland to an accessory gland and a secondary duct continues on, emptying into the base of the sheath opposite the proximal orifice of the fang. Venom then travels through the venom canal to the distal orifice on the rostral aspect near the tip of the fang. Fangs are shed periodically (6 to 10 weeks) and reserve fangs may be present, confusing the expected picture of two fang marks at a bite site.Conversely, only one functional fang may be present at a given time or the nature of the strike may produce a single fang injection. Viperid, elapid, and crotalid snakes also have rows of mandibular, maxillary, and palatine teeth that may puncture the skin. Vipers and pit vipers may envenomate from various positions. If grasped or stepped upon, the snake immediately twists to bite the hand or limb. This bite would be similar to an elapid bite. Venom may be injected by one or both fangs, at a depth determined by the length of the fang. A strike is a different maneuver; the snake positions itself into "S"-shaped folds, with the head slightly elevated. As the snake begins the strike, the mouth is opened to approximately a 160° angle, the fangs are erected to a 90° angle with the upper jaw, and the head is thrust forward toward the victim (Figure 3). The bite resulting from a strike of a viperid or crotalid snake is more like a hypodermic injection than a bite. The forward thrust of the curved fang on penetration of the skin may initiate closure of the fang, so that even a longfanged snake may make a subcutaneous rather than a deep, intramuscular injection. The depth of the injections and the amount of venom injected vary with the size and aggressiveness of the snake. The maximum striking distance is approximately two thirds the body length of the snake. Viperids and crotalids cannot propel the entire body through the air.

COLUBRID SNAKES
Venomous snakes in the family Colubridae have one to three elongated, fixed maxillary teeth (opisthioglyphous) at the caudal aspect of the dental arcade (rear-fanged) (Figure 1). Most snakes in this family are not considered dangerous to humans or animals, but human fatalities have been associated with the bite of the boomslang, Dispholidus typus, and illness has been produced by bites of the mangrove, Boiga dendrophila, and the California lyre snake, Trimorphodon vandenburghi. Colubrid venom is produced in a special gland called "Duvemoy's gland", the ducts of which empty into the mouth at the base of the fangs. The fangs are grooved on the rostral border and the venom is carried into the wound by capillary action during the chewing associated with the bites of these snakes.


Species response to snake envenomation is marked. Some of the factors involved are unknown, but immunity to the proteinaceous compounds in the venom may be an important protective mechanism. It has been assumed since antiquity that venomous snakes may be immune to their own venom. Pliny the Elder said, "The sting of the serpent is not aimed at the serpent."31 Modem technologies have established that neutralizing antibodies in the serum of the eastern diamondback rattlesnake (Crotalus adamanteus) protect the snake from accidental, self-inflicted envenomation. Serum from the rattlesnake is more effective than commercial antivenin for protecting mice from the effects of rattlesnake venom. The same is true of water moccasin (Agkistrodon piscivorus) serum against water moccasin venom. Similar results have been obtained from studies of the resistance of viperid and elapid snakes and the gila monster, Heloderma suspectum, to their own venom. It has been stated that the Australian tiger snake, Notechis scutatus, is 108,000 times more resistant to its own venom than is the guinea pig, Cavia porcellus. Such nonvenomous snakes as the king snake, Lampropeltis getulus, that prey upon other snakes and include crotalids in their diet are also resistant to crotalid venom. Knowledge of resistance to self-injected venom is important to the manager of captive collections of venomous snakes. It is not uncommon for a hyperexcited snake to bite itself during restraint procedures necessary for medical care. However, protection is not absolute, but is dependent upon the injected dose of venom.

Great diversity exists in mammalian resistance to snake venoms. Although the mongoose is highly adept at avoiding the slow strike of a cobra, it has also long been reputed to be resistant to cobra venom, and this has now been demonstrated in the laboratory.37 Nevertheless, a large venom dose may exceed the natural resistance and kill the mongoose if it makes a mistake and grasps a cobra too far caudal on the neck, allowing the snake to tum and bite. Other small carnivorous mammals that include snakes in their diet may also have natural resistance to the venom of snakes in their habitat. The Virginia opossum, Didelphis virginiana, is relatively resistant to crotalid venom.Likewise, the European hedgehog, Erinaceus europaeus, is capable of neutralizing some of the hemorrhagic factors in viper venom. Domestic and feral pigs (Sus scrofa) are also reputed to be resistant to crotalid venom, but the protection is provided by resistance of the thick skin to fang penetration and the layer of subcutaneous fat that retards absorption of the venom into the bloodstream. Intramuscular injection of venom may be lethal to the pig. Birds apparently have no immunity against snake venoms, even though raptorial birds may attack and kill venomous snakes. Feathers may prevent the fangs from penetrating the skin.

Some humans have claimed immunity to snakebite, particularly members of snake-worshipping cults and individuals who have been repeatedly bitten by venomous snakes. Solid evidence for long-term immunity is lacking. An Australian herpetologist allowed himself to be hyperimmunized and, at the end of a year of repeated injections, was receiving eight times the estimated lethal dose of tiger snake venom. At that time, his serum titer response was high, but a year later the titer had dropped and was likely unprotective. Attempts to vaccinate humans in high-risk areas have been less than satisfactory. In addition, individuals who have been repeatedly bitten by venomous snakes face the risk of anaphylaxis resulting from acquired sensitivity to the foreign proteins in the venom.