Chapter 26: Toxic Effects of Plants and Animals

History is replete with stories of the earliest humans using plant extracts and animal venoms for hunting, war, assassination, and political intrigue for millennia. Even the Ebers Papyrus, which dates to around 1550 bc, describes concoctions using plant substances as primary ingredients. The toxic properties of plants and animals often enhance their ability to survive. These toxic adaptations reflect how the organism interacts with its surroundings and with its predators. Some toxic compounds are used primarily to aid an animal in obtaining food while plants have developed toxic properties to specifically ward off being used as food. These toxic compounds are invaluable in the insight that they provide into the systems that they disrupt and poison. One major complication to the study of plant and animal poisons arises from their complexity as mixtures. Studies readily separate and evaluate individual components, but it is very difficult to use purified components to make the original toxin or venom. Nevertheless, extensive study of many toxins has contributed to a greater understanding of their biology and chemistry. Toxins have been utilized as tools to study human biochemistry and physiology in order to pave the way for new pharmaceuticals. In fact, some components are in active development for clinical use. Clinical evaluation of human poisoning is complicated by questionable identification of plant or animal species and the inability to quantify the level of exposure. In this chapter, an overview of specific plant and animal toxins and their effects will precede a short discussion of the considerable effort to harness natural pharmacopeia for clinical use.

The plant kingdom contains potentially 300,000 species, and the toxic effects of plants serve primarily as defense mechanisms against natural predators. Toxicity in humans can result from simply touching as well as ingesting plants to cause a truly wide array of deleterious effects. Toxic effects on humans can range from simple hay fever caused by exposure to plant pollen all the way to serious systemic reactions caused by ingestion of specific plants. Table 26-1 lists some of the poisoning syndromes that plants can produce (Nelson et al., 2007).

There are many variables that can affect the concentration of a plant’s toxin and that can be a major factor in the severity of reaction one will experience on exposure. These factors include what part of the plant exposure is from, the age of the plant, amount of sunlight and soil quality that the plant has grown in, and genetic differences within a species. Also, plant toxins fall under a number of different chemical structures, which is useful in understanding related toxins. Table 26-2 lists some of the common classifications.

The number and variety of plants that have some level of toxicity are far too many to be discussed here. Criteria for inclusion for discussion are based on how frequently human exposure occurs, the seriousness of these exposures, and also how well the toxic agent is scientifically understood. Excellent source material can be found in the comprehensive texts by Dart (2004), Cameron et al. (2009), Leikin and Paloucek (2008), and Nelson et al. (2007).

Skin

Irritant Contact Dermatitis

Plants that cause irritation of the skin on contact are rather common (Nelson et al., 2007). A list of such plants containing their family, genus, and common names can be found in Table 26-3. The trichomes, or barb-like hairs (Fig. 26-1), found on stinging nettles (Urtica species, Urticaceae) puncture skin on contact and release an irritating sap containing a mixture of formic acid, histamine, acetylcholine, and serotonin (Kavalali, 2003). Various species of Urtica can be found all over the world; however, the most dangerous is U. ferox (poisonous tree nettle), which is most common in New Zealand. Exposure has been reported to cause death in humans and animals. Mucuna pruriens (cowhage), which also deploys its toxin via barbed trichomes on contact, may cause pain, itching, erythema, and vesication. Mucinain, contained in the toxin, is the proteinase responsible for causing the pruritus (Southcott and Haegi, 1992).

Certain species of Ranunculus (buttercup) contain a compound known as ranunculin, which is enzymatically broken down into the toxin protoanemonin. On contact with skin, protoanemonin found in Anemone (buttercup) is converted to anemonin, which is the irritant directly responsible for the resulting dermatitis. If ingested, protoanemonin may cause severe irritation of the gastrointestinal tract (Kelch et al., 1992).

Damage to the stems or leaves of the genus Euphorbia (Euphorbiaceae, spurge family) causes exudation of a milky latex that contains diterpene esters that are irritating to the skin. Euphorbia marginata (snow-on-the-mountain) is a common plant in the United States that is used in flower arrangements by florists. Dermal contact with its latex can cause skin irritation (Urushibata and Kase, 1991). Also, serious eye irritation has been reported (Frohn et al., 1993). The poinsettia (Euphorbia pulcherrima, Fig. 26-2), which is ubiquitous at holiday times, may cause contact dermatitis (Massermanian, 1998).

Allergic Contact Dermatitis

Many people have experienced allergic dermatitis, most frequently from contact with poison ivy. Allergic dermatitis is an actual allergic reaction occurring within the skin as opposed to just a response to the presence of an irritant (Johnson et al., 1972). Due to this immunological component, the severity of the reaction can range widely.

Philodendron scandens (Araceae, arum family) and the toxicodendron group of plants, which contains Rhus radicans (poison ivy, Fig. 26-3), Rhus diversiloba (poison oak), and Rhus vernix (poison sumac), are all known to cause allergic dermatitis. P. scandens is a common houseplant that produces allergenic resorcinols, especially 5-n-heptadecatrienyl resorcinol (Knight, 1991). In the Rhus species the allergen is a fat-soluble substance called urushiol that can penetrate the stratum corneum where it then binds to Langerhans cells in the epidermis. These haptenated cells then migrate to lymph nodes, where T cells are activated resulting in the allergic response (Kalish and Johnson, 1990). Ingestion of Rhus species has been reported to cause generalized dermatitis (Oh et al., 2003). Sap of the mango fruit (Magnifera indica, Anacardiaceae) can also cause allergic dermatitis due to the presence of oleoresins that, with repeated exposure, will cross-react with allergens of poison ivy (Tucker and Swan, 1998).

Alkaloids present in the sap of daffodils, hyacinths, and tulips can sometimes cause irritation. Irritation can also be caused by contact with needle-like crystals of calcium oxalate, also known as raphides, which are present on these plants’ bulbs (Gude et al., 1988). The major culprit is the compound tulipalin-A, which causes “tulip fingers” from handling tulip bulbs (Christensen and Kristiansen, 1999). Tulipalin-A can be found in concentrations up to 2%. A safe threshold for this allergen is considered to be 0.01% (Hausen et al., 1983). Table 26-4 contains more plants that cause irritation due to oxalates.

“Latex-fruit syndrome” is the result of cross-sensitivity to latex in rubber gloves and some fruits. Hevea brasiliensis (the latex tree) produces prohevein, a chitin-binding polypeptide that is also found in several plants. Hevein, a 43–amino acid N-terminal fragment of prohevein, is the major binding component (Kolarich et al., 2005). Individuals who are allergic to rubber latex may become sensitized to fruits containing a chitinase with a hevein-like domain, such as banana, kiwi, tomato, and avocado (Blanco et al., 1999).

Lichens, such as species of Usnea and Cladonia, are known to cause dermatitis due to the production of usnic acid (a benzofuran) and related acids (Aalto-Korte et al., 2005). Usnic acid has also been implicated in hepatotoxicity following use of certain nonprescription weight loss supplements (Han et al., 2004).

Photosensitivity

Dermatitis does not necessarily have to be caused by skin contact. Consumption of Hypericum perforatum (St. John’s wort) by animals can lead to serious dermatitis and even may be life threatening. The toxic agent is hypericin (a bianthraquinone) that, once ingested and dispersed systemically, causes photosensitization of the animal’s skin. On exposure to sunlight, edematous lesions form on areas of skin that are not protected by hair such as the nose and ears (Sako et al., 1993). Photosensitization caused by St. John’s wort in humans is a rare occurrence; however, an increased response to therapeutic exposure to ultraviolet therapy has been reported (Beattie et al., 2005).

Respiratory Tract

Allergic Rhinitis

“Hay fever” or rhinitis from inhalation of plant pollens is a seasonal problem for many individuals. A chromosomal association in these individuals is under investigation (Blumenthal et al., 2006). Trees, grasses, and weeds are all responsible to contributing to airborne pollen. Grass species Poa and Festuca are major contributors along with pollen from several weed genera in the Asteraceae (eg, mugwort, Artemisia vulgaris, in Europe, and ragweed, Ambrosia sp., in North America, Fig. 26-4). The common denominator in the various pollen allergens is the conserved binding domain known as profiling, which is also found in birch pollen (Hirschwehr et al., 1998). Asthma and rhinitis have been linked to individuals who are exposed to cascara sagrada (Rhamnus purshiana) (Giavina-Bianchi et al., 1997), or workers in greenhouses in which bell peppers are growing (Groenewoud et al., 2006).

Cough Reflex

Workers who process peppers have a significantly increased incidence of coughing when specifically handling Capsicum annuum (sweet pepper) and Capsicum frutescens (red pepper). These two types of peppers produce the major irritants capsaicin (trans-8-methyl-N-vanillyl-6-nonenamide) and dihydrocapsaicin (Surh et al., 1998). Specific nerves in the airway have been found to be capsaicin-sensitive, which leads to the irritation and cough (Blanc et al., 1991).

Toxin-Associated Pneumonia

The pneumotoxin, 4-ipomeanol, is produced in sweet potato roots (Ipomea batatas, Convolvulaceae) by the mold Fusarium solani. 4-Ipomeanol is activated by human cytochrome P450s to an intermediate that binds to DNA (Alvarez-Diez and Zheng, 2004). In cattle and rabbits, the major P450 activator is CYP4B1 found in the lung that results in pneumonia. In the mouse, CYP4B1 is most abundant in the kidneys that results in renal toxicity and in humans multiple subsets of liver P450 enzymes are responsible for activating 4-ipomeanol (Baer et al., 2005).

Gastrointestinal System

Direct Irritant Effects

Ingestion of a toxic plant can cause irritation of the gastrointestinal tract often resulting in nausea, vomiting, and diarrhea. Many different compounds produced by plants can cause mild to severe versions of these effects. A list of plants known to cause these effects is provided in Table 26-5. Ingestion of ripe tung nuts (Aleurites fordii) causes abdominal pain, vomiting, and diarrhea. Outbreaks of poisoning are most likely to occur in children (Lin et al., 1996).

Toxic quinolizidine alkaloids are found in buffalo beans, also known as buffalo peas (Thermopsis rhombifolia), which grow naturally in the western United States. Ingestion by children causes nausea, vomiting, dizziness, and abdominal discomfort (McGrath-Hill and Vicas, 1997). Also, consumption by livestock of the mature plant with seeds has been reported to be fatal.

Nuts from Aesculus hippocastanum (horse chestnut, Fig. 26-5) and Aesculus glabra (Ohio buckeye) contain a glucoside called esculin. Ingestion by humans causes gastroenteritis, which increases in severity with the number of nuts consumed. Fortunately, esculin is poorly absorbed by the gastrointestinal tract of humans and its systemic effects are usually limited. However, in cattle, esculin may be hydrolyzed in the rumen resulting in release of the aglycone to cause systemic effects. This can lead to nervous system stimulation—marked by a stiff-legged gait and, in severe poisoning, tonic seizures with opisthotonus (Casteel et al., 1992). The triperpene saponin b-aescin in horse chestnut seed extract may have value in treatment of venous insufficiency in humans (Siebert et al., 2002).

Antimitotic Effects

Podophyllotoxin is found in Podophyllum peltatum (May apple, Berberidaceae) especially in its foliage and roots. In low doses, mild purgation occurs; however, overdose results in nausea and severe paroxysmal vomiting (Frasca et al., 1997). By binding microtubules, podophyllotoxin blocks mitosis from proceeding. This has made podophyllotoxin of interest for treatment of cancer (Schacter, 1996).

Found in the bulbs of Colchicum autumnale (autumn crocus, Liliaceae), colchicine blocks the formation of microtubules ultimately preventing successful mitosis. Ingestion of these bulbs causes severe gastroenteritis (nausea, vomiting, diarrhea, and dehydration). Severe poisoning can result in confusion, hematuria, neuropathy, renal failure and cardiotoxicity (Mendis, 1989).

Protein Synthesis Inhibition

The family Euphorbiaceae contains several genera that are known to be very toxic. The castor bean (Ricinus communis, Fig. 26-6) is an ornamental plant that produces seeds that, if eaten by children or adults, causes no symptoms of poisoning for several days after ingestion. Gradually, gastroenteritis develops resulting in some loss of appetite, with nausea, vomiting, and diarrhea. If a fatal dose is ingested, the gastroenteritis becomes extremely severe and is marked by persistent vomiting, bloody diarrhea, and icterus followed by death within six to eight days. A fatal dose for a child can be as few as five seeds and may be as low as 20 seeds for an adult. However, fatality occurs in less than 10% of ingestions when a “fatal” dose is consumed owing to the fact that the toxic protein is largely destroyed during digestion. The toxic agents are two lectins found in the beans: ricin I and ricin II of which ricin II is more toxic. Ricin II is made up of an A-chain and a B-chain. The B-chain is responsible for helping the A-chain get inside the cell. It binds to a terminal galactose residue on the cell membrane that then allows for the A-chain to be endocytosed (Wu et al., 2006). Once inside, the A-chain inactivates the 60s ribosomal subunit of cells by catalytic depurination of an adenosine residue within the 28s rRNA (Bantel et al., 1999), thereby blocking protein synthesis.

The seeds of Abrus precatorius (jequirity bean, Leguminosae) contain lectins known as abrins. Abrin-a, one of four isoabrins produced by the plant, has the highest inhibitory effect on protein synthesis and consists of an A-chain and a B-chain (Tahirov et al., 1994). Similar to ricin, the A-chain directly inhibits protein synthesis while the B-chain is responsible for getting the A-chain inside the cell (Ohba et al., 2004). The LD₅₀ of abrin when injected in mice is less than 0.1 μg/kg making abrin one of the most toxic substances known.

Plants that produce only A-chains are not nearly as toxic as those that pair them with a B-chain. Young shoots of pokeweed (Phytolacca americana, Phytolaccaceae, Fig. 26-7) can be ingested without toxicity; however, consumption of mature leaves and berries may cause nausea and diarrhea. The plant produces three isozymes of single-chain lectins that are capable of inhibiting protein synthesis in cells. However, these do not readily pass through a cells membrane but are capable of doing so with the help of a virus (Monzingo et al., 1993).

Wisteria floribunda (Leguminosae) is a common ornamental climbing vine that develops seeds in the fall. Ingestion of these seeds can cause severe gastroenteritis. Just a few seeds can produce headache, nausea, and diarrhea within hours, followed by dizziness, confusion, and hematemesis (Rondeau, 1993). The toxic agent is a lectin that binds N-acetylglucosamine on mammalian neurons.

Cardiovascular System

Cardioactive Glycosides

The best known cardioactive glycoside is Digitalis purpurea (foxglove, Scrophulariaceae, Fig. 26-8). However, others exist in the lily family, such as squill (Scilla maritima), which contains scillaren, and lily of the valley (Convallaria majalis), which contains convallatoxin in the bulbs, that have actions similar to digitalis. Also, milkweeds (Asclepias species, Asclepiadaceae) contain glycosides (Roy et al., 2005). Other cardiotoxic plants can be found in Table 26-6. The cardiac glycoside desglucouzarin in Asclepias asperula, like digitalis, inhibits Na⁺,K⁺-ATPase (Abbott et al., 1998). Two plants in the Apocynaceae (oleander family) also contain cardioactive glycosides. Thevetia peruviana (yellow oleander) is a common ornamental plant in the United States whose seeds contain the highest concentration of cardiac glycosides. The fatal dose to an adult is eight to 10 seeds (Prabhasankar et al., 1993). Clinical aspects of oleander toxicosis in sheep have been described (Aslani et al., 2004). Also, oleander poisoning may not be fatal (Pietsch et al., 2005).

Actions on Cardiac Nerves

Toxic alkaloids found in Veratrum viride (American hellebore, Liliaceae, Fig. 26-9), Veratrum album (European hellebore), and Veratrum californicum cause nausea, emesis, hypotension, and bradycardia on ingestion. Veratrum album has been used for centuries to “slow and soften the pulse.” The mixture of alkaloids includes protoveratrine, veratramine, and jervine that affects the heart by causing a repetitive response to a single stimulus resulting from prolongation of the sodium current (Jaffe et al., 1990). The bulbs of the wild camas (Zigadenus paniculatus and other species of Zigadenus, Liliaceae) contain Veratrum-like alkaloids (Peterson and Rasmussen, 2003).

Aconitum species, which have been used in western and eastern medicine for centuries, produce the toxic alkaloids aconitine, mesaconitine, and hypoaconitine. Poisoning may occur on ingestion but severity varies with the concentration of the alkaloids, which depends on species, place of origin, time of harvest, and processing procedures (Chan et al., 1994). Along with cardiac arrhythmias and hypotension, ingestion causes gastrointestinal upset and neurological symptoms. The alkaloids work by causing a prolonged sodium current with slowed repolarization in cardiac muscle (Peper and Trautwein, 1967) and in nerve fibers (Murai et al., 1990).

Grayanotoxins are produced exclusively by several genera of Ericaceae (heath family) and in particular they are found in Rhododendron ponticum (Fig. 26-10; Onat et al., 1991) R. macrophyllum (Casteel and Wagstaff, 1989), and Kalmia angustifolia (Burke and Doskotch, 1990). Ingestion of honey contaminated with grayanotoxins, brought there by bees, can produce a severe reaction called “mad honey poisoning.” The poisoning resembles aconitine poisoning in that there is bradycardia, hypotension, oral parasthesia, and gastrointestinal upset. Severe poisoning can result in respiratory depression and eventually loss of consciousness. Grayanotoxins bind to sodium channels in cardiac and muscle cells resulting in increased sodium conductance (Maejima et al., 2002).

Vasoactive Chemicals

American mistletoe (Phoradendron flavescens) and European mistletoe (Viscum album, Loranthaceae) are members of the same family and both produce a toxin that is marked for its effect on the cardiovascular system. Both phoratoxin (produced by American mistletoe) and viscotoxin (produced by European mistletoe) cause hypotension, vasoconstriction of the vessels in skin and skeletal muscle, and bradycardia resulting from negative inotropic actions on heart muscle. Phoratoxin is only one-fifth as active as the viscotoxins (Rosell and Samuelsson, 1988). Although serious poisoning from the plants is rare, it is possible to induce anaphylaxis with repeated injections of mistletoe extract (Bauer et al., 2005).

Ingestion of the fungus Claviceps purpurea (ergot), which grows on grains that are used for food, causes vasoconstriction. The “ergot gene cluster” is required for production of ergot alkaloids (Coyle and Panaccione, 2005; Haarmann et al., 2005). Ergot poisoning was called “St. Anthony’s fire” due to the blackened appearance of the limbs of some victims. In extreme cases, the vasoconstriction was severe enough that gangrene would develop in the extremities. Abortion in pregnant women is also common after ingestion of ergot-contaminated grains. Acremonium coenophialum, a fungus which grows on the grass tall fescue (Festuca arundinacea), produces some ergot alkaloids. Grazing cattle that ingest the contaminated grass develop “fescue toxicosis” (Blodgett, 2001; Hill et al., 1994). This condition results in decreased weight gain, decreased reproductive performance, and increased peripheral vasoconstriction. Pulmonary infection from Acremonium strictum has been noted in a horse (Pusterla et al., 2005).

Liver

Hepatocyte Damage

Pyrrolizidine alkaloids can be found in Senecio (groundsel, Asteraceae) and within four genera of Boraginaceae, Echium (bugloss), Cynoglossum (hound’s tongue), Heliotropium (heliotrope), and Symphytum (comfrey) Altamirano et al., 2005; Mei et al., 2005. Ingestion of significant concentrations of these alkaloids causes liver damage in the form of hepatic veno-occlusive disease associated with lipid peroxidation (Bondan et al., 2005). Cattle that graze on grass contaminated with Senecio have been found to develop hepatitis that can progress to death if allowed to continue grazing. Human deaths have also been reported in several countries and in Afghanistan, an epidemic of hepatic veno-occlusive disease arose from consumption of a wheat crop contaminated by Heliotropium (Tandon et al., 1978). The liver damage caused by ingestion clinically appears to be similar to cirrhosis and some hepatic tumors that can easily be mistaken to be the source of the disease (McDermott and Ridker, 1990). Consumption of these plants leads to a form of the Budd–Chiari syndrome, which is hallmarked by portal hypertension and obliteration of the small hepatic veins. Human consumption can occur from drinking “comfrey tea” that contains Symphytum (Rode, 2002). There are species differences in metabolism of the pyrrolizidine alkaloids (Huan et al., 1998).

Lantana camara (Verbenaceae), a shrub native to Jamaica, has been shown to poison livestock, particularly in India. Cattle grazing on the plant develop bile-related disorders including cholestasis and hyperbilirubinemia. Several triterpenoids have been isolated from the plant and in particular lantadene A (22-β-angeloyloxy-3-oxo-olean-12-en-28-oic acid) has been shown to be hepatotoxic (Sharma et al., 1991).

Mushroom Toxins

Most nonedible mushrooms may cause mild discomfort and are not life threatening; however, repeated ingestion of the false morel, Gyromitra esculenta, has been found to cause hepatitis (Michelot and Toth, 1991). The toxin gyromitrin is generally inactivated by boiling. Most fatal poisonings related to wild mushrooms are from ingestion of different species within Amanita, Galerina, and Lepiota (Karlson-Stiber and Persson, 2003). The dangerousness of Amanita phalloides (Fig. 26-11) and Amanita ocreata is why they are named “death cap” and “death angel,” respectively. Two types of toxins, phalloidin and amatoxins, can be found within A. phalloides. Phalloidin is capable of binding actin in muscle cells; however, it is not readily absorbed during digestion, which limits its harmful effects (Cappell and Hassan, 1992). Unfortunately, α-, β-, and γ-amanitins are readily absorbed due to being molecularly much smaller than phalloidin. Of the amatoxins, α-amanitin is the most toxic as it inhibits protein synthesis in hepatocytes by binding to RNA polymerase II (Jaeger et al., 1993). In addition to liver, intestinal mucosa and kidneys are also affected and serious clinical signs develop about three days after ingestion. In cases of severe poisoning, a liver transplant may be required. Amatoxin-a irreversibly inhibits acetylcholinesterase (Hyde and Carmichael, 1991).

Mycotoxins

Fumonisin toxins are produced by the fungus Fusarium that is known to grow on corn. Consumption of contaminated corn by horses leads to equine leukoencephalomalacia that is marked by lethargy, ataxia, convulsions, and ultimately death (Norred, 1993). The liver is the most affected organ in many species including horses, pigs, chickens, and rats (Riley et al., 1994). Ingestion in humans has been suggested to be associated with esophageal cancer (Yoshizawa et al., 1994). Fumonisins are diesters of propane-1,2,3-tricarboxylic acid and a pentahydroxyicosane containing a primary amino acid (Gurung et al., 1999) that is similar to sphingosine. This similarity is responsible for their toxicity as they block the enzymes involved in sphingolipid biosynthesis (Norred, 1993). In contrast, mycoestrogenic zearalenone induces CYP3A enzymes (Ding et al., 2006). Aflatoxin B1 has been shown to form guanine adducts and induce apoptosis in human hepatocytes (Reddy et al., 2006).

Kidney and Bladder

Carcinogens

The bracken fern (P. aquilinum), which is extremely common worldwide, is the only higher plant known to be carcinogenic in animals under natural feeding conditions. The commonest bladder tumors in cattle are epithelial and mesenchymal neoplasms (Kim and Lee, 1998). Ptaquiloside, a norsesquiterpene glucoside, is the known carcinogen present in the fern and it has been found to alkylate adenines and guanines of DNA (Rasmussen et al., 2003; Shakin et al., 1999). Bovine consumption of bracken fern has been shown to significantly increase chromosomal aberrations (Lioi et al., 2004). Also, evidence has been found that consumption of young bracken fern shoots by humans is associated with cancers of the mouth and throat (Alonso-Amelot and Avendano, 2002).

Kidney Tubular Degeneration

Species of Xanthium (cocklebur, Asteraceae) have been found to contain the toxin carboxyatractyloside, which causes microvascular hemorrhages in multiple organs (Turgut et al., 2005). Livestock poisoning has been noted to cause the outward signs of depression and dyspnea; however, internally the toxin causes tubular degeneration and necrosis in the kidney and centrilobular necrosis in the liver (del Carmen Mendez et al., 1998).

Consumption of the mushroom species Cortinarius has been found to cause acute renal failure but different species vary widely in toxicity and, therefore, edibility. In an investigation of a series of 135 poisonings related to Cortinarius ingestion, where death occurred in almost 15% of the cases, renal biopsy showed acute degenerative tubular lesions with inflammatory interstitial fibrosis (Bouget et al., 1990). Cortinarius orellanus and C. rubellus (Fig. 26-12) contain the deadly toxin orellanin, which triggers renal failure. Table 26-7 lists the common and scientific names and toxins for selective plants that are capable of inducing nephrotoxicity.

Blood and Bone Marrow

Anticoagulants

Fungal infections in sweet clover (Melilotus alba) have been found to produce dicumarol, a coumarin derivative that is a potent anticoagulant. Deaths in cattle have been reported and are caused by hemorrhages (Puschner et al., 1998).

Bone Marrow Genotoxicity

Argemone (Papaveraceae), a species of poppy, produces sanguinarine, a benzophenanthridine alkaloid that is known to intercalate DNA and have carcinogenic potential (Das et al., 2005). Studies in mice have shown that a single low dose of argemone oil increases chromosomal aberrations in bone marrow cells (Ansari et al., 2004).

Cyanogens

Cyanogens are found in a wide variety of plants including the kernels of apples, cherries, and peaches. The highest concentrations are found in the seeds of the bitter almond, Prunus amygdalus var amara. However, small children are susceptible to amygdalin poisoning if they consume enough peach (Prunus persica) kernels. Fortunately, the concentration present in seeds of apples is low enough that they are unlikely to cause a problem. Metabolism of amygdalin releases hydrocyanic acid that binds to the ferric ion in methemoglobin, which, if severe enough, results in cyanide poisoning with death from asphyxiation (Bromley et al., 2005; Jorgensen et al., 2005; Rosling, 1993). Severe cyanide poisoning can occur with so-called vitamin supplements (O’Brien et al., 2005).

Cassava produced from Manihot esculenta (Euphorbiaceae) is a major food source for some regions of Africa. The raw root contains a cyanogenic glucoside linamarin that is removed during processing of the root for human consumption. Unfortunately, local processing may be inadequate and that can lead to ingestion of linamarin. Chronic ingestion has been suggested to be the cause of epidemics of konzo, a form of tropical myelopathy with sudden onset of spastic paralysis (Tylleskar et al., 1992).

Nervous System

Epileptiform Seizures

The common and scientific names for selective plants that produce neurotoxins can be found in Table 26-8. Within the family Apiaceae, which contains carrots, the fleshy tubers of Cicuta maculata (water hemlock, Fig. 26-13) produce neurotoxic cicutoxin (a C17-polyacetylene). Consumption of a single tuber can result in fatal poisoning, characterized by tonic–clonic convulsions, owing to the cicutoxin binding to GABA-gated chloride channels (Uwai et al., 2000).

Members of the mint family (Labiatae) such as pennyroyal (Hedeoma), sage (Salvia), and hyssop (Hyssopus) are well known for their essential oils containing monoterpenes. Ingestion of these monoterpenes in concentrations much higher than those used for flavoring can cause tonic–clonic convulsions. In particular, menthol is a selective modulator of inhibitory ligand-gated channels (Hall et al., 2004).

Certain species within Strychnos (Loganiaceae) produce strychnine and brucine, which are known to cause increased CNS stimulation by blocking glycine-gated chloride channels. Strychnos nux vomixa, a small tree native to India, produces seeds that have been implicated in cases of unintentional poisoning (Wang et al., 2004).

Excitatory Amino Acids

Red algae (Digenia simplex) under certain conditions can proliferate rapidly leading to the notorious beach vacating “red tide” and producing kainic acid. Kainic acid may be ingested by humans who eat filter-feeding mussels that have eaten red algae. Acute symptoms are most notably gastrointestinal distress, headache, hemiparesis, confusion, and seizures. Severe exposure can result in severe memory deficits and sensorimotor neuropathy (Teitelbaum et al., 1990). Isodomoic acid from Nitzschia sp. has seizure-inducing properties (Kotaki et al., 2005).

The fungi Amanita muscaria (fly agaric, Fig. 26-14) and Amanita pantherian (panther agaric) produce the excitatory amino acid ibotenic acid (isoxazole amino acid) and its derivative muscimol that is neurotoxic (Li and Oberlies, 2005). Poisoning produces central nervous system depression, ataxia, hysteria, and hallucinations. Myoclonic twitching and seizures sometimes develop (Benjamin, 1992). Other genera of fungi have been marked for their hallucinogenic actions, notably Psilocybe, which contains psilocin and psilocybin (Tsujikawa et al., 2003).

Willardiine, an agonist on glutamate receptors, has been isolated from Acacia willardiana, Acacia lemmoni, Acacia millefolia, and Mimosa asperata (Gmelin, 1961) and causes excitation of the nervous system. Seeds from the legume Lathyrus sativus (grass pea) also contain an excitatory amino acid known as β-N-oxalyl-l-α,β-diaminopropionic acid (Warren et al., 2004). Consumption of seeds over long periods can cause lathyrism to develop. Affected individuals have corticospinal motor neuron degeneration with severe spastic muscle weakness and atrophy but little sensory involvement (Spencer et al., 1986). Thiocyanate from linamarin can stimulate neuronal glutamate receptors, leading to degeneration of corticospinal motor pathways (Spencer, 1999).

Motor Neuron Demyelination

Karwinskia humboldtiana is a shrub found in the southwestern United States, Mexico, and Central America that produces anthracenones in its seeds. Both human and livestock poisonings have been known to occur (Bermundez et al., 1986). Several days following ingestion, ascending flaccid paralysis develops with demyelination of large motor neurons in the legs and eventually leads to bulbar paralysis in fatal cases (Martinez et al., 1998). In addition to neurotoxicity, the anthracenones in Karwinskia, especially peroxisomicine A₂, cause lung atelectasis, emphysema, and massive liver necrosis. Inhibition of catalase in peroxisomes has been proposed as the mechanism of cell toxicity (Martinez et al., 1997).

Cerebellar Neurons

The legumes Swainsonia cansescens, Astragalus lentiginosus (spotted locoweed), and Oxytropis sericea (locoweed) produce a toxic indolizidine alkaloid called swainsonine. These weeds can be accidentally consumed by grazing cattle causing aberrant behavior with hyperexcitability and locomotor difficulty (James et al., 1991). In fatal cases there is cytoplasmic foamy vacuolation of cerebellar neurons. Swainsonine causes marked inhibition of liver lysosomal and cytosomal α-mannosidase and Golgi mannosidase II resulting in abnormal accumulation of brain glycoproteins and mannose-rich oligosaccharides that ultimately causes cell death (Tulsiani et al., 1988). Embellisia fungi from locoweed produce a locoweed-like toxicosis (McLain-Romero et al., 2004).

Parasympathetic Stimulation

Certain mushrooms of the genera Inocybe, Clitocybe, and Omphalatus contain significant amounts of muscarine, the principal neurotransmitter in the parasympathetic nervous system. Consumption of one these species results in extreme parasympathetic activation resulting in diarrhea, sweating, salivation, and lacrimation (de Haro et al., 1999).

Parasympathetic Block

Atropine, l-hyoscyamine, and scopolamine are belladonna alkaloids that can be found in varying concentrations in several genera of Solanaceae such as Datura stramonium (jimson weed, Fig. 26-15), Hyoscyamus niger (henbane), Atropa belladonna (deadly nightshade, Fig. 26-16), and Duboisia myoporoides (pituri). These alkaloids all effectively block the muscarinic receptor, essentially turning off the parasympathetic drive at the target organ. This explains why tachycardia, dry mouth, dilated pupils, and decreased gastrointestinal motility all occur on ingestion of these toxins (Smith et al., 1991). Consumption of grains contaminated with seeds from Datura sp produce poisoning typical of belladonna alkaloids (van Muers et al., 1992). Of the three alkaloids, scopolamine is the most potent; however, sizable doses of l-hyoscyamine or atropine can produce similar effects. Large doses can cause confusion, bizarre behavior, hallucinations, and subsequent amnesia and in severe intoxication, tachycardia may be completely absent (Caksen et al., 2003).

The seeds of Solanum dulcamara (bittersweet), which are used in flower arrangements, contain the glycoalkaloid solanine that on ingestion causes tachycardia, dilated pupils, and hot and dry skin—symptoms similar to atropine poisoning (Ceha et al., 1997).

Sensory Neuron Block

Capsaicin found in C. annuum (sweet pepper) and C. frutescens (red pepper) causes a burning sensation on vanilloid-type (VR1) sensory receptors. It also desensitizes the transient potential vanilloid 1 receptor (TRPV1) of sensory endings of C-fiber nociceptors to stimuli, a property which has therapeutic use in treating chronic pain (Szalcsany, 2002). Capsaisin also can relax ileal smooth muscle (Fujimoto et al., 2006). Fortunately, desensitization produced by capsaicin is not due to cell death (Dedov et al., 2001). Polygodial, a sesquiterpene found in Polygonum hydropiper, also desensitizes the TRPV1 (Andre et al., 2006), whereas resiniferatoxin activates TRVP1 (Raisinghani et al., 2005). Pyrethum from chrysanthemum flowers blocks sodium channels in the insect nervous system (McGovern and Bakley, 1999).

Skeletal Muscle and Neuromuscular Junction

Neuromuscular Junction

Anabasine, an isomer of nicotine, is present in Nicotiana glauca (tree tobacco, Solanaceae) and produces prolonged depolarization of the junction after a period of excessive stimulation. Consumption of N. glauca leaves can result in flexor muscle spasm and gastrointestinal irritation, followed by severe, generalized weakness, and respiratory compromise (Mellick et al., 1999). Curare, which is used as a poison placed on the tips of arrows, is also a potent neuromuscular blocking agent and is obtained from tropical species of Strychnos toxifera and Chondrodendron tomentosum. Anabaena flosaquae, a species of alga, can produce under the right conditions a neurotoxin anatoxin A that is known to be fatal. Anatoxin A, ingested by animals that drink pond water with the alga present, depolarizes and blocks the animal’s nicotinic and muscarinic acetylcholine receptors, which can cause death from respiratory arrest within minutes to hours (Short and Edwards, 1990).

Delphinium barbeyi (tall larkspur, Ranunculaceae), which grows naturally in the western United States, contains methyllycaconitine. Ingestion of the toxin by cattle results in muscle tremors and ataxia followed by prostration and can ultimately lead to respiratory arrest in fatal cases. Methyllycaconitine is similar to curare in that it has a high affinity for the acetylcholine receptor at the neuromuscular junction. Physostigmine has been used successfully as an antagonist to treat some cases of methyllycaconitine poisoning (Pfister et al., 1994).

Skeletal Muscle Damage

Certain species of Thermopsis produce seeds that contain quinolizidine alkaloids. Human poisoning from eating the seeds is rare, but cases have been reported in young children who experienced abdominal cramps, nausea, vomiting, and headache lasting up to 24 hours (Spoerke et al., 1988). Livestock grazing on Thermopsis montana (false lupine, mountain goldenbanner, Fig. 26-17) develop locomotor depression and recumbency due to areas of necrosis in skeletal muscle that have been found on autopsy (Keeler and Baker, 1990).

Consumption of Cassia obtusifolia (sicklepod, Leguminosae) seeds by livestock causes a degenerative myopathy in cardiac and skeletal muscle. Extracts of C. obtusifolia have been found to inhibit NADH-oxidoreductase in bovine and swine mitochondria in vitro (Lewis and Shibamoto, 1989).

White snakeroot (Ageratina altissima, Asteraceae, Fig. 26-18), a common plant in the central and western United States, can be accidentally eaten by grazing cattle. On ingestion, the cattle exhibits tremors and humans who drink the milk of an affected cow can get “milk sickness” (Beier et al., 1993). The toxic effects are attributed to tremetone, a benzofuran, which blocks gluconeogenesis from lactate, resulting in acidosis, tremor, and ultimately death (Polya, 2003).

Bone and Tissue Calcification

Bone and Soft Tissue

Consumption of Solanum malacoxylon (Solanaceae) by sheep and cows can cause a marked decrease in bone calcium and calcification of the entire vascular system due to the presence of a water-soluble vitamin D–like substance. In severe cases other organs can also be affected such as the lungs, joint cartilage, and kidney.

Consumption of Cestrum diurnum (day-blooming jasmine, Solanaceae) and Cestrum laevigatum has been found to cause hypercalcemia and soft tissue calcification in grazing cattle (Durand et al., 1999) and chickens (Mello and Habermehl, 1992), which is due to the presence of a dihydroxyvitamin D3 glycoside in the leaves (Durand et al., 1999). Hay that has been contaminated with C. laevigatum caused a marked centrilobular and midzonal hepatic necrosis in goats (Peixato et al., 2000).

Reproduction and Teratogenesis

Abortifacients

Besides its actions on the nervous system, swainsonine, the active alkaloid in the legumes Astragalus and Oxytropus, also causes abortions in pregnant livestock that accidentally ingest locoweeds (Bunch et al., 1992).

Foliage and seeds of Leucaena leucocephala, Leucaena glauca, and Mimosa pudica contain a toxic amino acid, mimosine, which on ingestion by cattle leads to uncoordinated gait, goiter, and reproductive disturbances including infertility and fetal death (Kulp et al., 1996). Mimosine has been found to arrest the cell cycle in late G1 phase that helps explain its toxic effects (Perry et al., 2005).

Lectins present in bitter melon seeds (Momordica charantia, Curcurbitaceae) have antifertility, abortifacient, and embryotoxic actions on ingestion. Components of the lectins known as momorcharins are known to induce midterm abortion in humans (Wang and Ng, 1998).

Caulophyllum thalictroides (blue cohosh, Berberidaceae) contains a toxin known as caulophylline that is teratogenic in rats (Kennelly et al., 1999) and herbal preparations of blue cohosh have been used to terminate pregnancy (Jones and Lawson, 1998). In fact, nicotine toxicity may result from blue cohosh use as an abortifacient (Rao and Hofman, 2002).

Teratogens

Ingestion of V. californicum (California false hellebore, Liliaceae, Fig. 26-19) by pregnant sheep is known to cause malformations in its offspring that can include cyclopia, exencephaly, and microphthalmia. As with most teratogens, the severity of malformations depends on what developmental stage the fetus is in at the time of exposure. Limb defects are common with exposure during the fourth to fifth week of gestation; fetal stenosis of the trachea will occur with ingestion on days 31 to 33 (Omnell et al., 1990). Many other species of animals are susceptible to V. californicum poisoning including cows, goats, chickens, rabbits, rats, and mice (Omnell et al., 1990), hamsters (Gaffield and Keeler, 1993), lambs (Keeler, 1990), and rainbow trout embryos (Crawford and Kocan, 1993). The toxic alkaloid called jervine causes teratogenesis by blocking cholesterol synthesis that, among other things, prevents a proper response of fetal target tissue to the sonic hedgehog gene (Shh). Shh has an important role in proper developmental patterning of head and brain, and blocking cholesterol synthesis has been shown experimentally to cause a loss of midline facial structures (Cooper et al., 1998).

Pregnant cattle grazing on Lupinus caudatus and Lupinus formosus (lupines, Leguminosae), N. glauca (tree tobacco, Solanaceae), and Conium maculatum (poison hemlock, Solanaceae) have been found to experience a myriad of fetal deformations if ingestion occurs during a sensitive gestational period. The toxic alkaloids in these plants are anagyrine (L. caudatus), ammodendrine (L. formosus), anabasine (N. glauca), and coniine (C. maculatum, Forsyth et al., 1996). It is thought that these alkaloids depress fetal movements that can lead to malformations (Lopez et al., 1999).

Recent research concerning the use of plant toxins in a clinical setting has blossomed (Ball and Kowdley, 2005). A number of factors make plant toxins of particular interest including their diverse effects, availability, and cost. With a growing group of people who wish to return to a more “natural” way of life, a medicine that is derived straight from a plant is much more marketable than one that is completely chemically synthesized. For example, root extract of the African Uzara plant has been used as an antidiarrheal remedy for centuries; however, its mechanism of action was only speculative. Recent research has shown that Uzara root extract reduced chloride secretion by the gut specifically by inhibiting Na⁺,K⁺-ATPase. This effect was seen even in the presence of cholera toxin that causes potent diarrhea by increasing chloride secretion in the gut (Schulzke et al., 2011). Interestingly, anemonin, which is the active skin irritant produced by species of Ranunculus (buttercup), has been found to show potent anti-inflammation effects under certain conditions. The compound was found to reduce nitric oxide production that resulted in a lessened inflammatory response to inflammatory stimuli (Lee et al., 2008). This mechanism could provide a new anti-inflammatory treatment modality and is being tested for possible medical use. Silymarin and usuic acid may have promise as anticancer agents (Mayer et al., 2005a,b). Finally, seed extract from M. pruriens is used by native Nigerians as a pretreatment for snakebites from Naja sputatrix (Javan spitting cobra). When this was tested in rats, the seed extract helped protect against the cardiorespiratory and neuromuscular depressant effects of the snake venom (Fung et al., 2011). Thus, old herbal remedies are a ripe field of study for many of their effects can be beneficial yet toxic at high enough concentrations. A goal for new research is to elucidate the mechanism of action so that treatments can be tailored to the individual needs and toxic effects can be avoided or interactions with conventional drugs can be minimized (Izzo and Ernst, 2001). As our knowledge of plant toxins and their mechanisms of action expands, the number of new and clinically useful plant remedies and medical products is bound to increase. Technological advances in chemical purification and identification will permit better toxic characterization as well (Arilla et al., 2006).

Toxic chemicals produced by plants can cause something as innocuous as a mild rash on the skin all the way to death by respiratory arrest. It is important to remember that these toxins are first and foremost defense mechanisms against natural predators. However, we benefit greatly from plants as many have produced potent antibiotics and pharmacologic therapies for myriad diseases that afflict humans.

The animal kingdom consists of more than 100,000 species spread through major phyla including arthropods, mollusks, chordates, etc (Mebs, 2002). Venomous animals are capable of producing a poison in a highly developed exocrine gland or group of cells and can deliver their toxin during a biting or stinging act. The venom is the sum of all natural venomous substances produced in the animal (Ménez et al., 2006). Conversely, poisonous animals have no specific mechanism or structure for the delivery of their poisons, and poisoning usually takes place through ingestion. Venomous or poisonous animals are widely distributed throughout the animal kingdom, from the unicellular protistan Alexandrium (Gonyaulax) to certain mammals, including the platypus and the short-tailed shrew. At least 400 species of snakes are considered dangerous to humans. Myriad venomous and poisonous arthropods exist, and toxic marine animals are found in almost every sea and ocean (Russell and Nagabhushanam, 1996; Mackessy, 2010; Mebs, 2002).

Animal venom may play a role in offense (as in the capture and digestion of food), in the animal’s defense (as in protection against predators or aggressors), or in both functions. Venoms used in an offensive posture are generally associated with the oral pole, as in the snakes and spiders, while those used in a defensive function are usually associated with the aboral pole or with spines, as in the stingrays and scorpion fishes. In the snake, the venom provides a food-getting mechanism. Its secondary function is its defensive status. In contrast, in venomous spiders, toxin is used to paralyze the prey before the extraction of hemolymph and body fluids. The venom is not primarily designed to kill the prey, but it is only to immobilize the organism for feeding. The same can be said for scorpions, although they do use their venom in defense. In fishes, such as the scorpion fishes and stone fishes, and in elasmobranches, such as the stingray, the venom apparatus is generally used in the animal’s defense. Poisonous animals, on the other hand, usually derive their toxins through the food chain. As such, poison is often a metabolite produced by microorganisms, plants, or animals. Poisons are sometimes concentrated as they pass through the food chain from one animal to another. It is also worth noting that animal and plant toxins tend to differ significantly in their chemical complexity, yet both are capable of causing massive harm. Plant toxins tend to be smaller compounds or proteins and often times a single offending substance can be pinpointed. Conversely, animal toxins must be studied in the context of the entire venom or poison that typically is very complex and contains many individual toxic compounds and very large proteins that essentially work together to cause their effects. Just the same as the preceding section on plant toxins, certain criteria were employed to effectively narrow down the animal toxins discussed. Specifically considered were how often does human exposure occur, how significant is an exposure, and in general how much is known about the animal’s toxic properties.

Venoms are very complex, containing polypeptides, high- and low-molecular-weight proteins, amines, lipids, steroids, aminopolysaccharides, quinones, glucosides, and free amino acids, as well as serotonin, histamine, and other substances. Some venoms are known to consist of more than a hundred proteins. The venom is a source of peptides and proteins that act on myriad exogenous targets such as ion channels, receptors, and enzymes within cells and on cell membranes (Ménez et al., 2006). Studying venom is important for several reasons. First, venoms offer valuable insight into the systems they act on such as the cardiovascular system, nervous system, coagulation, and homeostasis. Second, venoms are useful as a source of potential new drugs, with at least five agents already on the market and dozens undergoing preclinical or clinical trials (Menez et al., 2005). Third, a greater understanding of venoms favors development of improved protection against envenomation (Ménez et al., 2006).

Novel instrument developments have permitted the greater application of mass spectrometry, coupled with various separation technologies, to tease out the complexity of natural venoms, thereby identifying the peptide and protein components of venoms (Escoubas, 2006). The technology allows considerable resolution of extremely small amounts of venom. Fig. 26-20 demonstrates the application of gel filtration and high-pressure liquid chromatography (HPLC), as cone snail venom was fractionated into numerous peptides with varying activities (Olivera et al., 1990). Similar fractionations have been performed on many other types of venom to identify the individual components. Unfortunately, studying the chemistry, pharmacology, and toxicology of venoms requires isolating and dismantling the venoms and losing the synergy among multiple components. Nevertheless, advanced technology will permit peptide sequencing, and the characterization of posttranslational modifications, such as glycosylation, and the discovery of new pharmacophores. Most venoms probably exert their effects on almost every cell and tissue, and their principal pharmacologic properties are usually determined by the amount of a fraction that accumulates at an activity site. Table 26-9 reveals the extremely large range in the LD₅₀ of different toxic compounds and venoms injected intravenously into mice.

The bioavailability of a venom is determined by its composition, molecular size, amount or concentration gradient, solubility, degree of ionization, and the rate of blood flow into that tissue, as well as the properties of the engulfing surface itself. The venom can be absorbed by active or passive transport, facilitated diffusion, or pinocytosis, among other physiologic mechanisms. Besides the bloodstream, the lymph circulation not only carries surplus interstitial fluid produced by the venom but also transports larger molecular components and other particulates back to the bloodstream. Thus, the larger toxins of snake venoms, particularly those of Viperidae, probably enter the lymphatic network preferentially and then are transported to the central venous system in the neck (Russell, 2001). Because lymphatic capillaries, unlike blood capillaries, lack a basement membrane and have fibroelastic “anchoring filaments,” they can readily adjust their shape and size, facilitating absorption of excess interstitial fluid along with macromolecules contained in a venom.

The site of action and metabolism of venom is dependent on its diffusion and partitioning along the gradient between the plasma and the tissues where the components are deposited. Once the toxin reaches a particular site, its entry to that site is dependent on the rate of blood flow into that tissue, the mass of the structure, and the partition characteristics of the toxin between the blood and the particular tissue. Receptor sites appear to have highly variable degrees of sensitivity. In the case of complex venoms, there may be several, if not many, receptor sites. There is also considerable variability in the sensitivity of those sites for the different components of a venom.

A venom may also be metabolized in several or many different tissues before undergoing excretion. Some venom components are metabolized distant to the receptor site(s) and may never reach the primary receptor in a quantity sufficient to affect that site. The amount of a toxin that tissues can metabolize without endangering the organisms may also vary. Organs or tissues may contain enzymes that catalyze a host of reactions, including deleterious ones. Once a venom component is metabolically altered, the end substance is excreted primarily through the kidneys. The intestines play a minor role, and the contributions by the lungs and biliary system have not been determined. Excretion may be complicated by the direct action of the venom on the kidneys themselves.

There are more than one million species of arthropods, generally divided into 25 orders, of which only about 10 orders are of significant venomous or poisonous importance. These include the arachnids (scorpions, spiders, whip scorpions, solpugids, mites, and ticks), the myriapods (centipedes and millipedes), the insects (water bugs, assassin bugs, and wheel bugs), beetles (blister beetles), Lepidoptera (butterflies, moths, and caterpillars), and Hymenoptera (ants, bees, and wasps). Several texts and papers that deal with venomous and poisonous arthropods are available (Bettini, 1978; Pick, 1986; Cohen and Quistad, 1998; Russell, 2001; Kuhn-Nentwig, 2003; Isbister et al., 2004).

The number of deaths from arthropod stings and bites is unknown. In Mexico, parts of Central and South America, North Africa, and India, deaths from scorpion stings, for instance, exceed several thousand a year. Spider bites probably do not account for more than 200 deaths a year worldwide. A common problem faced by physicians in suspected spider bites relates to the differential diagnosis. The arthropods most frequently involved in the misdiagnoses were ticks (including their embedded mouthparts), mites, bedbugs, fleas (infected flea bites), Lepidoptera insects, flies, vesicating beetles, water bugs, and various stinging Hymenoptera. Among the disease states that were confused with spider or arthropod bites or stings were erythema chronicum migrans, erythema nodosum, periarteritis nodosum, pyroderma gangrenosum, kerion cell-mediated response to a fungus, Stevens–Johnson syndrome, toxic epidermal necrolysis, herpes simplex, and purpura fulminans. Any arthropod may bite or sting and not eject venom. Finally, some arthropod venom poisonings accentuate the symptoms and signs of an existing undiagnosed subclinical disease that further complicates diagnosis.

Scorpions

Of the more than 1000 species of scorpions, the stings of more than 75 can be considered of sufficient importance to warrant medical attention (Keegan, 1980; Polis, 1990; Russell, 2001). Some of the more important scorpions are noted along with their location in Table 26-10. In addition, members of the genera Pandinus, Hadrurus, Vejovis, Nebo, and some of the others are capable of inflicting painful and often erythematous lesions.

Many scorpion venoms contain low-molecular-weight proteins, peptides, amino acids, nucleotides, and salts, among other components (Possani et al., 2000; Goldin, 2001; Rodriguez de la Vega and Possani, 2004, 2005). The neurotoxic fractions are generally classified on the basis of their molecular size; the short-chain toxins are composed of 20 to 40 amino acid residues with three or four disulfide bonds and appear to affect potassium or chloride channels, while the long-chain toxins have 58 to 76 amino acid residues (6500–8500 Da) with four disulfide bonds and affect mainly the sodium channels (Mouhat et al., 2004). These particular toxins may have an effect on both voltage-dependent channels. The amino acid content is known for more than 90 species, and there appears to be a high number of cysteine residues in most of these venoms. The toxins can selectively bind to a specific channel of excitable cells, thus impairing the initial depolarization of the action potential in the nerve and muscle that results in their neurotoxicity. It appears that the way some scorpion venoms differently affect mammalian, as opposed to insect tissues, is related to the structural basis of the gates in the two organisms. Not all scorpions, however, have fractions that affect neuromuscular transmission. Recently, Rodriguez de la Vega and Possani (2005) constructed a phylogenetic tree using 191 different amino acid sequences from long-chain peptides and discussed their functional divergence and extant biodiversity. The effects of scorpion venom on various potassium channels have been reviewed (Rodriguez de la Vega and Possani, 2004).

The symptoms and signs of scorpion envenomation differ considerably depending on the species (Russell, 2001). Common offenders are members of the family Vejovidae, generally found in the southwestern and western United States, Central America, and South America. Their sting gives rise to localized pain, swelling, tenderness, and mild parasthesia. Systemic reactions are rare, although weakness, fever, and muscle fasciculations have been reported.

Envenomations by some members of the genus Centruroides are clinically the most important, particularly in the western United States. The bark scorpion, C. exilicauda, is often found hiding under the loose bark of trees or in dead trees or logs, and may frequent human dwellings. Straw to yellowish-brown or reddish-brown in color, it is often easily distinguishable from other scorpions in the same habitat by its long, thin telson, or tail, and its thin pedipalps, or pincer-like claws. In children, their sting may produce initial pain, although some children do not complain of pain and are unaware of the injury. The area becomes sensitive to touch, and merely pressing lightly over the injury will elicit an immediate retraction. Usually there is little or no local swelling and only mild erythema. The child becomes tense and restless and shows abnormal and random head and neck movements. Often the child will display roving eye movements. Centruroides vittatus, the striped bark scorpion (Fig. 26-21), is commonly involved in envenomenation but fatalities are rare. In their review of Centruroides sculpturatus stings, Rimsza et al. (1980) noted visual signs, including nystagmus roving eye and oculogyric movements, in 12 of 24 patients stung by this scorpion. Tachycardia is usually evident within 45 minutes as well as some hypertension. Although this is not seen in children as early or as severely as in adults, it is often present within an hour following the sting. Respiratory and heart rates are increased, and by 90 minutes the child may appear quite ill. Fasciculations may be seen over the face or large muscle masses, and the child may complain of generalized weakness and display some ataxia or motor weakness. Opisthotonos is not uncommon. The respiratory distress may proceed to respiratory paralysis. Excessive salivation is often present and may further impair respiratory function. Slurring of speech may be present, and convulsions may occur. If death does not occur, the child usually becomes asymptomatic within 36 to 48 hours.

In adults the clinical picture is somewhat similar, but there are some differences (Russell, 2001). Almost all adults complain of immediate pain after the sting, regardless of the Centruroides species involved. Adults do not show the restlessness that is seen in children. Instead, they are tense and anxious. They develop tachycardia and hypertension, and respirations are increased. They may complain of difficulties in focusing and swallowing. In some cases, there is some general weakness and pain on moving the injured extremity. Convulsions are very rare, but ataxia and muscle incoordination may occur. Most adults are asymptomatic within 12 hours, but may complain of generalized weakness for 24 hours or more.

Spiders

Of the 30,000 or so species, at least 200 have been implicated in significant bites on humans. Spiders are predaceous, polyphagous arachnids that generally feed on insects or other arthropods. Additional information on spider bites can be found elsewhere (Gertsch, 1979; Maretiĉ and Lebez, 1979; Russell, 2001). Table 26-11 provides a short list of spiders with their associated toxins and the targets of their toxins.

All spiders except the Uloboridae family possess a venom apparatus that produces neurotoxins designed to paralyze or kill prey. Spider venoms are complex mixtures of low-molecular-weight components, including inorganic ions and salts, free acids, glucose-free amino acids, biogenic amines and neurotransmitters, and polypeptide toxins. The acylpolyamines, composed of a hydrophobic aromatic carboxylic acid linked to a lateral chain of one to nine aminopropyl, aminobutyl, or aminopentyl units, are voltage-dependent open-channel blockers (sodium, calcium, and potassium channels) and/or blockers of the ion channel associated with glutamate receptors. They also act on nicotinic acetylcholine receptors. The acylpolyamines possess insecticidal activity and induce fast insect paralysis via a reversible block of the insect neuromuscular junction. Polypeptide toxins include the ion channel blockers, pore-forming peptides, and enzymes. In particular, hanatoxins 1 and 2 have allowed characterization of voltage-dependent potassium channels (Swartz and MacKinnon, 1995; Yellen, 2002). The ω-agatoxins have been demonstrated to block voltage-sensitive vertebrate calcium channels (Corzo and Escoubas, 2003). The ω-atracotoxins have greater selectivity on insect voltage-sensitive calcium channels (Wang et al., 2001). Many more spiders and the spider peptide toxins have been studied to date (Fatehi et al., 1997; Liu et al., 1997; Corzo and Escoubas, 2003; Adams, 2004; Kuhn-Nentwig et al., 2004; Rodriguez de la Vega and Possani, 2004; Ushkaryov et al., 2004; Wilson and Alewood, 2006). The small spider peptide toxins are relatively easy to produce by chemical synthesis or by recombinant means. Peptide toxins from spiders have proved useful in discriminating between different cellular components of native ion channel currents and for the molecular isolation and designation of cellular receptors (Escoubas et al., 2000; Corzo and Escoubas, 2003; Adams, 2004).

Poor case definition and spider identification contribute to the controversy regarding the clinical consequences and inappropriate diagnosis of spider bites worldwide. Many patients never see the spider, yet many skin lesions or areas of necrosis are attributed to spider bites. The medically important spiders include the Agelenopsis sp., Latrodectus sp., Loxosceles sp., Atrax sp., Hadronyche sp., and Phoneutria sp. Table 26-12, summarized from Isbister and White (2004), lists local and systemic effects for known cases of major spider groups in Australia.

Agelenopsis Species (American Funnel Web Spiders)

The American funnel web spider (Agelenopsis aperta) contains three classes of agatoxins that target ion channels (Adams, 2004). The α-agatoxins appear to be use-dependent, noncompetitive antagonists of the glutamate receptor channels. These are low-molecular-weight acylpolyamines that are devoid of amino acids. Mass spectrometry analysis has identified more than 33 α-agatoxins in A. aperta venom (Chesnov et al., 2001). The μ-agatoxins are 36 to 37 amino acid, C-terminal amidated, peptides with four internal disulfide bridges (Skinner et al., 1989). These μ-agatoxins cause increased spontaneous release of neurotransmitter from presynaptic terminals and repetitive action potentials in motor neurons. In addition, the μ-agatoxins are specific for insect sodium channels. The ω-amatoxins are a structurally diverse group of peptides that are selective for voltage-activated calcium channels. There are four types of ω-amatoxins that can be distinguished by sequence similarity and their spectrum of action against insect and vertebrate calcium channels. The action of the α-agatoxins is synergized by the μ-agatoxins causing channels to open at the normal resting potentials. It is interesting to note that the agatoxins are used as selective pharmacologic probes to characterize ion channels in organs such as brain and heart, and have been evaluated as candidate biopesticides.

Latrodectus Species (Widow Spiders)

Found throughout the world in all continents with temperate or tropical climates, these spiders are commonly known as the black widow, brown widow, or red-legged spider. They, however, have many other common names in English: hourglass, poison lady, deadly spider, red-bottom spider, T-spider, gray lady spider, and shoe-button spider. Although both male and female widow spiders are venomous, only the female has fangs that are large and strong enough to penetrate the human skin. Mature Latrodectus mactans females range in body length from 10 to 18 mm, whereas males range from 3 to 5 mm (Fig. 26-22). These spiders have a globose abdomen varying in color from gray to brown to black, depending on the species. In the black widow, the abdomen is shiny black with a red hourglass or red spots and sometimes with white spots on the venter (Russell, 2001).

The latrotoxins, a family of high-molecular-weight proteins that are found in Latrodectus venoms, target different classes of animals including vertebrates, insects, and crustaceans (Grishin, 1998, 1999; Ushkaryov et al., 2004). The toxins are synthesized as large precursors containing around 1000 amino acid residues (around 132–156 kDa) that undergo proteolytic processing to 110 to 130 kDa and activation in the lumen of the venom gland. Mature latrotoxins are structurally conserved and contain multiple ankyrin repeats. At least seven different latrotoxins have been isolated and all are large acidic proteins (pI ~5.0–6.0). Some are called latroinsectotoxins because they affect insects but not vertebrates, and one protein called α-latrocrustatoxin is active only in crayfish. All latrotoxins stimulate massive release of neurotransmitters after binding to specific neuronal receptors.

α-Latrotoxin is the most studied protein that is toxic only to vertebrates and not to insects or crustaceans (Ushkaryov et al., 2004). It is a presynaptic toxin that is said to exert its toxic effects on the vertebrate central nervous system depolarizing neurons by increasing intracellular [Ca²⁺] and by stimulating exocytosis of neurotransmitters from nerve terminals (Holz and Habener, 1998). The purported three-dimensional structure of α-latrotoxin consists of tetrameric complexes with a central channel that inserts into the lipid bilayer. A G-protein-coupled receptor latrophilin and a single-transmembrane receptor neurenin are high-affinity binding sites for α-latrotoxin (Ushkaryov et al., 2004). The latrotoxins act by both calcium-dependent and calcium-independent mechanisms. In fact, a mutant recombinant α-latrotoxin that does not form pores has been invaluable in furthering our understanding of the multiple actions of the toxin (Volynski et al., 2003). α-Latrotoxin and its mutants are versatile tools for the study of exocytosis. In particular, studies with this toxin have helped confirm the vesicular hypothesis of transmitter release (Hurlbut et al., 1990), establish the requirement of calcium ion for endocytosis (Ceccarelli and Hurlbut, 1980), characterize individual neurotransmitter sites in the central nervous system (Auger and Marty, 1997), and identify two families of important neuronal cell surface receptors (Krasnoperov et al., 1997).

Bites by the black widow are described as sharp and pinprick-like, followed by a dull, occasionally numbing pain in the affected extremity and by pain and cramps in one or several of the large muscle masses (Russell, 2001). Rarely is there any local skin reaction except during the first 60 minutes following the bite. Muscle fasciculations frequently can be seen within 30 minutes of the bite. Sweating is common, and the patient may complain of weakness and pain in the regional lymph nodes, which are often tender on palpation and occasionally are enlarged; lymphadenitis is frequently observed. Pain in the low back, thighs, or abdomen is a common complaint, and rigidity of the abdominal muscles is seen in most cases in which envenomation has been severe. Severe paroxysmal muscle cramps may occur, and arthralgia has been reported. Hypertension is a common finding, particularly in the elderly after moderate to severe envenomations. Blood studies are usually normal.

Loxosceles Species (Brown or Violin Spiders)

These primitive spiders are variously known in North America as the fiddle-back spider or the brown recluse (Fig. 26-23). There are over 100 species of Loxosceles. The abdomen of these spiders varies in color from grayish to orange and reddish-brown to blackish and is distinct from the pale yellow to reddish-brown background of the cephalothorax. This spider has six eyes grouped in three dyads. Females average 8 to 12 mm in body length, whereas males average 6 to 10 mm. Both males and females are venomous (Russell, 2001).

The venom of Loxosceles spiders appears to contain phospholipase, protease, esterase, collagenase, hyaluronidase, deoxyribonuclease, ribonuclease, dipeptides, dermonecrosis factors, and sphingomyelinase D. The venom has coagulation and vasoconstriction properties and it causes selective vascular endothelial damage. There are adhesions of neutrophils to the capillary wall with sequestration and activation of passing neutrophils by the perturbed endothelial cells (Patel et al., 1994). In Loxosceles intermedia, the toxic effects appear to be associated with a 35-kDa protein that demonstrates a complement-dependent hemolytic activity and a dermonecrotic-inducing factor. ³¹P-Nuclear magnetic resonance assay of the four bands representing proteins, measuring 34 kDa in the venom, produced three proteins with sphingomyelinase D activity (Merchant et al., 1998). An endotoxemic-like shock, showing eosinophilic material in the proximal and distal tubules and tubular necrosis, was the most common histopathological finding, preceded in mice by prostration, acute cachexia, hypothermia, neurological changes, and hemoglobinuria (Tambourgi et al., 1998).

The bite of this spider produces about the same degree of pain as does the sting of an ant, but sometimes the patient may be unaware of the bite. In most cases, a local burning sensation, which may last for 30 to 60 minutes, develops around the injury. Pruritus over the area often occurs, and the area becomes red, with a small blanched area surrounding the reddened bite site. Skin temperature usually is elevated over the lesion area. With significant envenomations, the reddened area enlarges and becomes purplish during the subsequent one to eight hours. It often becomes irregular in shape, and as time passes, hemorrhages may develop throughout the area. A small bleb or vesicle may form at the bite site, increase in size, and rupture with subsequent pustule formation. The red hemorrhagic area continues to enlarge, as does the pustule. The whole area may become swollen and painful, and lymphadenopathy is common. During the early stages, the lesion often takes on a bull’s-eye appearance, with a central white vesicle surrounded by the reddened area and ringed by a whitish or bluish border. The central pustule ruptures, and necrosis to various depths can be visualized (Russell, 2001).

In serious bites, the lesion can measure 8 × 10 cm² with severe necrosis invading muscle tissue. On the face, large lesions resulting in extensive tissue destruction and requiring subsequent plastic surgery sometimes are seen after bites by Loxosceles laeta in South America. Systemic symptoms and signs include fever, malaise, stomach cramps, nausea and vomiting, jaundice, spleen enlargement, hemolysis, hematuria, and thrombocytopenia. Fatal cases, while rare, usually are preceded by intravascular hemolysis, hemolytic anemia, thrombocytopenia, hemoglobinuria, and renal failure (Russell, 2001).

Steatoda Species

These spiders are variously known as the false black widow, combfooted, cobweb, or cupboard spiders. The female of Steatoda grossa differs from L. mactans and Latrodectus hesperus in having a purplish-brown abdomen rather than a black one. It is less shiny, and its abdomen is more oval than round. It may have pale yellow or whitish markings on the dorsum of the abdomen, and no markings on the venter. The abdomen of some species is orange, brown, or chestnut in color, and often bears a light band across the anterior dorsum (Russell, 2001).

The venom of Steatoda paykulliana stimulates the release of transmitter substances similar to Latrodectus venom (Cavalieri et al., 1987). The venom is said to form ionic channels permeable for bivalent and monovalent cations, and the duration of time in the open state depends on the membrane potential (Sokolov et al., 1984). S. paykulliana venom induces strong motor unrest, clonic cramps, exhaustion, ataxia, and then paralysis in guinea pigs. Bites by S. grossa or Steatoda fulva in the United States have been followed by local pain, often severe; induration; pruritus; and the occasional breakdown of tissue at the bite site (Russell, 2001).

Cheiracanthium Species (Running Spiders)

The 160 species of this genus have an almost circumglobal distribution, although only four or five species have been implicated in bites on humans (Russell, 2001). Cheiracanthium punctorium, Cheiracanthium inclusum, Cheiracanthium mildei, Cheiracanthium diversum, and Cheiracanthium japonicum are often implicated in envenomations. The abdomen is convex and egg shaped and varies in color from yellow, green, or greenish-white to reddish-brown; the cephalothorax is usually slightly darker than the abdomen. The chelicerae are strong, and the legs are long, hairy, and delicate. The spider ranges in length from 7 to 16 mm. Like Phidippus jumping spiders but even more so, Cheiracanthium tends to be tenacious and sometimes must be removed from the bite area. For that reason there is a high degree of identification following the bite of these spiders. The most toxic venom fraction is said to be a protein of 60 kDa, and the venom is high in norepinephrine and serotonin.

The patient usually describes the bite by C. inclusum as sharp and painful, with the pain increasing during the first 30 to 45 hours. The patient complains of dull pain over the injured part. A reddened wheal with a hyperemic border develops. Small petechiae may appear near the center of the wheal. Skin temperature over the lesion is often elevated, but body temperature is usually normal. Lymphadenitis and lymphadenopathy may develop. C. japonicum produces more severe manifestations, including severe local pain, nausea and vomiting, headache, chest discomfort, severe pruritus, and shock (Russell, 2001).

Theraphosidae Species (Tarantulas)

True tarantulas are members of the family Theraphosidae and there are around 800 species that are distributed worldwide, but especially in tropical or semitropical regions. Tarantulas are predators and they feed on various vertebrate and invertebrate preys that are captured after envenomation with venoms that act rapidly and irreversibly on the central and peripheral nervous systems. In humans, reported bites elicit mild to severe local pain, strong itching, and tenderness that may last for several hours. Edema, erythema, joint stiffness, swollen limbs, burning feelings, and cramps are common. In more severe cases, strong cramps and muscular spasms lasting up to several hours may be observed. Poecilotheria and Stromatopelma sp. appear to be most toxic to humans, although no fatalities have been reported (Escoubas and Rash, 2004).

Tarantula venoms have been extensively studied, and modern purification often uses orthogonal HPLC separations that combine reversed-phase and ion-exchange chromatography. Most spider peptide toxins appear to have basic pI in the nine to 11 range (Escoubas et al., 2000). At least 33 peptide toxins have been described from various tarantula venoms. These have a molecular weight of 3000 to 5700 Da, and targets include voltage-gated potassium, sodium, and calcium channels, tetrodotoxin-sensitive channels, and acid-sensing ion channels, which are sensitive to extracellular pH (reviewed by Escoubas and Rash, 2004). At least two distinct structural motifs have been characterized: the inhibitory cystine knot (ICK) with a consensus sequence of C_IX_3-7–C_IIX_3-8–C_IIIX_0-7–C_IVX_1-4–C_VX_4-13–C_VI and disulfide bond pairing of C_I–C_IV, C_II–C_V, and C_III–C_VI (Craik et al., 2001), and the disulfide-directed β-hairpin (DDH) motif that comprises an antiparallel β-hairpin stabilized by two disulfide bridges (Wang et al., 2000). Tarantula toxins have been classified as long-loop ICK, short-loop ICK, or DDH toxins (Escoubas and Rash, 2004).

Theraphosid spiders contain several toxins that are being evaluated for development as antiarrhythmic or as antinociceptive drugs. In particular, Grammostola mechanotoxin 4 from Grammostola spatulata has considerable promise as an antiarrhythmic. Protoxin I and II from Thrixopelma pruriens have promise as analgesics because they inhibit the tetrodotoxin-resistant sodium channels (Middleton et al., 2002).

Future work on the toxins of tarantulas will encompass genomic and proteomic approaches. Expanding knowledge suggests trends in the association of primary and three-dimensional structures with pharmacologic activity. Continued improvement in isolation and purification technologies plus the combination of cDNA library screening with mass spectrometry will prove invaluable in characterization of toxin function and definition of toxin targets.

Ticks

Many of the approximately 900 species of ticks are associated with disease in humans and wild and domesticated animals (Rash and Hodgson, 2002; Barker and Murrell, 2004; Steen et al., 2006). Tick paralysis is caused by the saliva of certain ticks of the families Ixodidae, Argasidae, and Nuttalliellidae. The tick bite involves insertion of cutting, tube-like mouthparts through the host’s skin with anchoring so that the tick can feed for hours, days, or weeks. Saliva from the salivarium flows outward initially and the blood meal flows inward afterward. Ticks are known to transmit the organisms causing Lyme disease, Rocky Mountain spotted fever, babesiosis, leptospirosis, Q fever, ehrlichiosis, typhus, tick-borne encephalitis, and others. In fact, the import of immunosuppression by tick saliva in the transmission of flaviviruses has been discussed (Nuttall and Labuda, 2003).

Tick saliva contains a number of active constituents (Steen et al., 2006). For example, saliva from Ixodes scapularis contains apyrase (ATP-diphosphohydrolase), which hydrolyzes ADP that is released at the bite site thereby inhibiting ADP-induced platelet aggregation (Mans et al., 1998); kininase (ACE-like protein or angiotensin-converting enzyme-like protein), which hydrolyzes circulating kinins and reduces the host inflammatory response (Francischetti et al., 2003); glutathione peroxidase (Das et al., 2001); serine protease inhibitors, which inhibit coagulation enzymes (Valenzuela, 2004); an anticomplement protein that inhibits an enzyme in the alternative pathway for complement (Valenzuela et al., 2000); an amine-binding protein that binds serotonin, histamine, and other biogenic amines (Sangamnetdej et al., 2002); and prostanoids (PGE₂ and PGF_2α) (Inokuma et al., 1994). A discussion of toxins from other species may be found elsewhere (Cavassani et al., 2005; Steen et al., 2006).

Potentially 50 species of ticks are associated with clinical paralysis (Russell, 2001). As tick bites are often not felt, the first evidence of envenomation may not appear until several days later, when small macules 3 to 4 mm in diameter develop that are surrounded by erythema and swelling, often displaying a hyperemic halo. The patient often complains of difficulty with gait, followed by paresis and eventually locomotor paresis and paralysis. Problems in speech and respiration may ensue and lead to respiratory paralysis if the tick is not removed. The saliva of Ixodes holocyclus has yielded a peptide holocyclotoxin-1 that may cause paralysis (Masina and Broady, 1999). Peak paralytic activity was found between 60 and 100 kDa, and was a trimer of a neurotoxic protein subunit of 23 kDa (Crause et al., 1993). Symptoms resolve rapidly on removal of the tick (Russell, 2001).

These elongated, many-segmented brownish-yellow arthropods have a pair of walking legs on most segments, and they are fast moving, secretive, and nocturnal and are found worldwide. They feed on other arthropods and even small vertebrates and birds. The first pair of legs behind the head is modified into poison jaws. Centipedes range in length from 3 to almost 300 mm. In the United States, the prevalent biting genus is a Scolopendra species. The venom is concentrated within the intracellular granules, discharged into vacuoles of the cytoplasm of the secretory cells, and moved by exocytosis into the lumen of the gland; from thence ducts carry the venom to the jaws (Ménez et al., 1990).

Centipede venoms contain high-molecular-weight proteins, proteinases, esterases, 5-hydroxytryptamine, histamine, lipids, and polysaccharides (Mebs, 2002). Such venom contains a heat-labile cardiotoxic protein of 60 kDa that produces, in humans, changes associated with acetylcholine release (Gomes et al., 1983). The bite produces two tiny punctures, sharp pain, immediate bleeding, redness, and swelling often lasting for 24 hours. Localized tissue changes and necrosis have been reported, and severe envenomations may cause nausea and vomiting, changes in heart rate, vertigo, and headache. In the most severe cases, there can be mental disturbances (Bush et al., 2001; Russell, 2001; Mebs, 2002).

Ranging in length from 20 to 300 mm, these arthropods are cylindrical, worm-like creatures, mahogany to dark brown or black in color, and bearing two pairs of jointed legs per segment. In Australia and New Guinea particularly, the repellent secretions expelled from the sides of their bodies contain a toxin of benzoquinone derivatives plus a variety of complex substances such as iodine and hydrocyanic acid, which the animal makes use of to produce hydrogen cyanide (Kuwahara et al., 2002). Some species can spray these defensive secretions, and eye injuries are not uncommon. The lesions produced by millipedes consist of a burning or prickling sensation and development of a yellowish or brown-purple lesion; subsequently, a blister containing serosanguinous fluid forms, which may rupture. Eye contact can cause acute conjunctivitis, periorbital edema, keratosis, and much pain; such an injury must be treated immediately (Russell, 2001).

Heteroptera (True Bugs)

The clinically most important of the true bugs are the Reduviidae (the reduviids): the kissing bug, assassin bug, wheel bug, or cone-nose bug of the genus Triatoma (Russell, 2001). Generally, they are parasites of rodents and common in the nests of wood rat or in wood piles. These are elongated bugs with freely movable, cone-shaped heads, and straight beaks. The most commonly involved species appear to be Triatoma protracta, Triatoma rubida, Triatoma magista, Reduvius personatus, and Arilus cristatus. The average length of these bugs is 19 mm. The venom of these bugs appears to have apyrase activity and to lack 5-nucleotidase, inorganic pyrophosphatase, phosphatase, and adenylate kinase activities, but it is fairly rich in protease properties. It inhibits collagen-induced platelet aggregation. Three peptides isolated from the saliva of predatory reduviids are 34 to 36 amino acid residues in size, are calcium channel blockers similar to ω-conotoxins, and belong to the four-loop disulfide bridge scaffold structural class (Corzo et al., 2001). The bites of Triatoma species are painful and give rise to erythema, pruritus, increased temperature in the bitten part, localized swelling, and—in those allergic to the saliva—systemic reactions such as nausea and vomiting and angioedema. With some bites the wound area will slough, leaving a depression.

The water-dwelling true bugs are of at least three families, Naucoridae, Belostomatidae, and Notonectidae, which are capable of biting and envenomating humans (Russell, 2001). They are found in lakes, ponds, marshes, quiet fresh water, and swimming pools. Lethocerus americanus, a Belostomatidae, ranges in length from 12 to 70 mm, but some water bugs may reach 150 mm. The dorsal side is usually tan or brown, but it may be brightly colored, while the ventral side is brown. They are very strong insects and can immobilize snails, tadpoles, salamanders, and even small fish and water snakes. Water bug saliva is said to contain digestive enzymes, neurotoxic components, and hemolytic fractions. ApoLp-III isolated from the hemolymph of Lathocerus medius is about 19 kDa and has an amino acid composition high in methionine. If molested, water bugs will bite, and their bites give rise to immediate pain, some localized swelling, and possibly induration and formation of a small papule.

Hymenoptera (Ants, Bees, Wasps, and Hornets)

Formicidae (Ants)

Most ant species sink their powerful mandibles into the flesh, providing leverage, and then drive their stings into the victim. Most ants have stings, but those that lack them can spray a defensive secretion from the tip of the gaster, which is often placed in the wound of the bite. Ants of the different species vary considerably in length, ranging from less than 1.5 to over 35 mm. Clinically important stinging ants are the harvesting ants (Pagonomyrmex), fire ants (Solenopsis), and little fire ants (Ochetomyrmex). The harvester ants are large red, dark brown, or black ranging in size from 6 to 10 mm and having fringes of long hairs on the posterior of their heads (Russell, 2001).

The venoms of the ants vary considerably. The venoms of the Ponerinae, Ecitoninae, and Pseudomyrmex are proteinaceous in character. The Myrmicinae venoms are a mixture of amines, enzymes, and proteinaceous materials, histamine, hyaluronidase, phospholipase A, and hemolysins, which hemolyze erythrocytes and mast cells. Formicinae ant venom contains about 60% formic acid. Fire ant venoms are poor in polypeptides and proteins, but are rich in alkaloids such as solenopsine (Russell, 2001; Mebs, 2002). The sting of the fire ant gives rise to a painful burning sensation, after which a wheal and localized erythema develop, leading in a few hours to a clear vesicle. Within 12 to 24 hours, the fluid becomes purulent and the lesion turns into a pustule. It may break down or become a crust or fibrotic nodule. In multiple stings there may be nausea, vomiting, vertigo, increased perspiration, respiratory difficulties, cyanosis, coma, and even death. Cross-exposure to the venom of other species of ants is possible. Allergic reactions and fatal anaphylactic shock are seen in sensitized victims (Hoffman, 2006). Treatment of ant stings is dependent on their number, whether an allergic reaction is involved, and whether there are possible complications.

Apidae (Bees)

This family includes the bumble bees, honeybees, carpenter bees, and yellow jackets. The commonest stinging bees are Apis mellifera and the Africanized bee, Apis mellifera adansonii, and the incidence of Hymenoptera poisonings is increasing. The venom of the Africanized bee is not remarkably different from that of the European bee, A. m. mellifer (Fig. 26-24). The former bee is smaller and gives less venom, but its aggressiveness is such that attacks of 50 to hundreds of bees are not unusual (Russell, 2001).

The venom contains biologically active peptides, such as melittin, apamine, mast cell–degranulating peptide, and others, as well as phospholipases A₂ and B, hyaluronidase, histamine, dopamine, monosaccharides, and lipids (Mebs, 2002). Melittin, which is secreted as the 70 amino acid prepromelittin, consists of 26 amino acids with no cysteines that have natural detergent-like properties and causes erythrocyte lysis. Melittin also forms tetramers with pores, thereby facilitating ion transport through membranes. In particular, melittin tetramers cause a breakdown of the resting potential and rapid depolarization of nociceptors, which induces pain (Demsey, 1990; Bechinger, 1997). The compound apamine contains 18 amino acids cross-linked by two disulfide bridges. Apamine is a blocker of calcium-dependent potassium channels and is thought to be the “lethal factor” (Habermann, 1984). In addition to apamine, mast cell–degranulating peptide is also a basic peptide containing 22 amino acids with two disulfide bonds. Besides stimulating release of histamine, this peptide specifically inhibits voltage-dependent potassium channels (Dreyer, 1990; Baku, 1999).

Bee stings typically produce immediate, sharp or burning pain, slight local erythema, and edema followed by itching. The edema may vary depending on location of the sting. It is said that 50 stings can be serious and lead to respiratory dysfunction, intravascular hemolysis, hypertension, myocardial damage, hepatic changes, shock, and renal failure. With 100 or more stings, death can occur. In patients who are allergic to bee stings, immediate allergic reaction with the risk of anaphylactic shock requires urgent medical treatment (Mebs, 2002; Hoffman, 2006).

Vespidae (Wasps)

This family includes wasps and hornets. These venoms contain a high content of peptides, which include mastoparan in wasps and hornets and crabolin from hornet venom. These peptides release histamine from mast cells and consist of 13 to 17 amino acids with no disulfide bridges. Other peptides named wasp kinins cause immediate pain, vasodilation, and increased vascular permeability leading to edema. These venoms also contain phospholipases and hyaluronidases, which contribute to the breakdown of membranes and connective tissue to facilitate diffusion of the venom. These proteins also contribute to the allergenicity of the venoms (Mebs, 2002; Hoffman, 2006).

Lepidoptera (Caterpillars, Moths, and Butterflies)

The urticating hairs, or setae, of caterpillars are effective defensive weapons that protect some species from predators. The setae are attached to unicellular poison glands at the base of each hair. Both the larvae and the adults are capable of stinging, either by direct contact with the setae or indirectly when the creature becomes irritated. It appears that contraction of the caterpillar’s abdominal muscles is sufficient to release the barbs from their sockets, allowing them to become airborne. The toxic material found in the venom glands contains aristolochic acids, cardenolides, kallikrein, and histamine among other substances. Fibrinolytic activity has been found in 16 and 18 kDa components (isoelectric point of 8.5); coagulation defects such as prolonged prothrombin and partial thromboplastin times have been detected, and decreases in fibrinogen and plasminogen have been noted. It is thought that the hemorrhagic syndrome cannot be classified as being either totally fibrinolytic or a syndrome such as disseminated intravascular coagulopathy. The spicules of Thaumetopoea pityocampa contain a 28-kDa toxin called thaumetopoein, which is a strong dermal irritant and highly allergenic peptide (Kawamoto and Kumada, 1984; Russell, 2001; Mebs, 2002).

In some parts of the world the stings of several species of Lepidoptera give rise to a bleeding diathesis, often severe and sometimes fatal. Envenomation by members of the family Saturniidae, the buck moths, the grapeleaf skeletonizer (family Zygaenidae), the puss moth (family Megalopygidae), and the brown-tailed moth (Euproctis species) generally gives rise to little more than immediate localized itching and pain, usually described as burning, followed in some cases by urticaria, edema, and occasionally fever. The hemolymph and spicules contain highly active clotting enzymes, a protease with fibrinolytic activity and an enzyme that activates prothrombin. In the more severe cases—often due to Megalopyge, Dioptidae, Automeris, and Hemileuca species—there is localized pain as well as papules (sometimes hemorrhagic) and hematomas; on occasions there may also be headache, nausea, vomiting, hematuria, lymphadenitis, and lymphadenopathy. Contacts with larvae of the saturnid moths in South America (Lonomia acheolus and Lonomia oblique, Fig. 26-25) can cause severe coagulopathy, due to inhibition of clotting factor XIII by a venom component called lonomine V. Severe envenomation can cause cerebral hemorrhage and death (Guerrero et al., 1999).

Human interest in this group of mollusks has been due to the beautiful patterns on their shells. Cone snails were known to Roman scholars and natural history collectors, as the shells were often made into jewelry. The first record of fatality from cone snail sting may be found in the book of Rumphius from 1705. The genus Conus is a group of approximately 500 species of carnivorous predators found in marine habitats that use venom as a weapon for prey capture (Fig. 26-26). Cone snails have a venom duct for synthesis and storage of venom and hollow harpoon-like teeth for injection of the venom (Rockel et al., 1995).

Cone snails may be divided into three groups depending on preferred prey. The largest group contains worm-hunting species that feed on polychaetes (segmented marine worms in the phylum Annelida). The second group is molluscivorous and hunts other gastropods. The final group is piscivorous and has venoms that rapidly immobilize fish (Olivera, 1997, 2002; Mebs and Kauferstein, 2005).

There are probably over 100 different venom components per species (Terlau and Olivera, 2004). Components have become known as conotoxins, which may be rich in disulfide bonds, and conopeptides. Molecular targets include G-protein-coupled receptors, neuromuscular transporters, and ligand- or voltage-gated ion channels. Some components have enzymatic activity.

Fig. 26-27 provides an overview of peptidic Conus venom components, indicating gene superfamilies, disulfide bond characteristics, and general targets (McIntosh et al., 1999; Olivera, 2002). The two major divisions of Conus toxins are the disulfide-rich conotoxins and the peptides that lack multiple disulfide cross-links. The arrangement of cysteines in the primary sequence is restricted to only a few patterns, which may be diagnostic of the gene superfamily. Most disulfide-rich conotoxins are small and consist of 12 to 30 amino acids. These toxins contain an unusually diverse complement of posttranslationally modified amino acids, including hydroxyproline, O-glycosylated serine or threonine, γ-carboxyl-glutamate, sulfated tyrosine, and d-amino acids (Craig et al., 1999). Apparently, the posttranslational modification enzyme γ-carboxylase is present in their venom ducts (Bandyopadhyay et al., 2002). The enzyme has a recognition signal in the pro region of the precursor that instructs the enzyme to modify specific amino acid residues in the mature toxin region. Thus, the pro region of conopeptide precursors provides potential anchor-binding sites for posttranslational modification enzymes (Hooper et al., 2000).

Cone snails could be called sophisticated practitioners of combination drug therapy. After injection, multiple conopeptides act synergistically to affect the targeted prey. The term toxin cabal has been applied to this coordinated action of the conopeptide mixture. The fish-hunting species Conus purpurascens apparently has two distinct cabals whose effects differ in time and space. The “lightning-strike cabal” causes immediate immobilization of the injected prey because various venom components inhibit voltage-gated sodium channel inactivation and block potassium channels, resulting in massive depolarization of axons in the vicinity of the injection site and a tetanic state. The second physiologic cabal, the “motor cabal,” acts more slowly as conotoxins must be distributed throughout the body of the prey. The overall result is total inhibition of neuromuscular transmission. Various conopeptides inhibit presynaptic calcium channels that control neurotransmitter release, the postsynaptic neuromuscular nicotinic receptors, and the sodium channels involved in the muscle action potential (Terlau et al., 1996).

Conopeptides can affect many ion channels (Olivera et al., 1994; Terlau and Olivera, 2004). For example, μ-conotoxins are peptides with 22 to 25 amino acids and six cysteine residues that block sodium currents by acting at a site that overlaps the tetrodotoxin-sensitive site of sodium channels. The main effect of the unusually hydrophobic δ-conotoxins, which are peptides with an inhibitory cysteine knot motif pattern of disulfide bridges, is the inhibition of the fast inactivation of sodium currents, which affects the shape and duration of the action potential. This can lead to a massive electrical hyperexcitation of the complete organism. The κ-, κA-, and κM-conotoxins target potassium channels. The ω-conotoxins are peptides that target calcium channels, and these toxins have been widely used in neuroscience research. A characteristic feature of the ω-conotoxins is their high content of basic amino acids. One peptide, ω-conotoxin MVIIA from Conus magus, has been developed as ziconotide (Prialt^®) for the treatment of intractable pain (Olivera, 2000). Other conotoxins are being studied as potential antinociceptive agents.

Conopeptides also target ligand-gated ion channels that mediate fast synaptic transmission (Kandel et al., 2000). One major group of ligand-gated ion channels is activated by acetylcholine, serotonin, γ-aminobutyric acid, or glycine. These proteins differ in ligand specificity and selectivity for the permeant ion. The second family activates the glutamate receptors, N-methyl-d-aspartate and kainate/AMPA receptors. A third group activates synaptic transmission at certain synapses via the ATP receptors. It is evident from Fig. 26-27 that there are conopeptides that target these three different families of ligand-gated ion channels. It is clear that our understanding of the structure and function of conopeptides is rapidly expanding and additional information regarding these toxins is available in an excellent review (Terlau and Olivera, 2004).

Lizards

The Gila monster (Heloderma suspectum, Fig. 26-28) and the beaded lizards (Heloderma horridum) are divided into five subspecies. These large, corpulent, relatively slow moving, and largely nocturnal reptiles have few enemies other than humans. They are far less dangerous than is generally believed. Their venom is transferred from venom glands in the lower jaw through ducts that discharge their contents near the base of the larger teeth of the lower jaw. The venom is then drawn up along grooves in the teeth by capillary action (Brown and Carmony, 1991). The venom of this lizard has serotonin, amine oxidase, phospholipase A, a bradykinin-releasing substance, helodermin, gilatoxin, and low-proteolytic as well as high-hyaluronidase activities, but lacks phosphomonoesterase and phosphodiesterase, acetylcholinesterase, nucleotidase, ATPase, deoxyribonuclease, ribonuclease, amino acid oxidase, and fibrinogenocoagulase activities. The clinical presentation of a helodermatid bite can include pain, edema, hypotension, nausea, vomiting, weakness, and diaphoresis. No antivenin is commercially available. Treatment is supportive (Strimple et al., 1997).

The venom has been shown to contain a 25-kDa protein, helothermine, containing 223 amino acids and four pairs of disulfide bonds, which appears to inhibit Ca²⁺ flux from the sarcoplasmic reticulum (Morrissette et al., 1995; Nobile et al., 1996). A fraction causing hemorrhage in internal organs and the eye, a glycoprotein of 210 amino acid residues with plasma kallikrein-like properties, has also been described (Datta and Tu, 1997). A 35 amino acid residue, helodermin produces hypotension that is partially attributed to activation of glibenclamide-sensitive K⁺ channels (Horikawa et al., 1998). In fact, helospectin I and II and helodermin are nonamidated, vasoactive intestinal peptide-like peptides, isolated from the salivary gland venom of the lizards H. suspectum and H. horridum, which have strong and potent vasodilator effects (Uddman et al., 1999).

Snakes

General Information and Classification

Snakes have a three-chambered heart and rely almost exclusively on an enlarged right lung (which spans approximately half of the body length) for respiration. Of the approximately 2700 known species of snakes, about 20% are considered to be venomous (Mebs, 2002; Mackessy, 2010). Venomous snakes primarily belong to the following families: Viperidae (vipers), Elapidae, Atractaspididae, and Colubridae. The vipers are further divided into subfamilies, an example of which is the Crotalinae, or pit vipers, which possess a pit between the eyes and nostrils that serves as a heat sensor to detect warm-blooded animals. Some of the subfamilies are regarded as separate families altogether depending on the classification scheme. Overall the Colubridae are considered the largest venomous family, and are composed of nearly 60% of all snakes. The Atractaspididae family is known for burrowing into the ground and possessing the ability to expose their fangs without opening their mouth.

Another characteristic that is often used for self-defense or hunting prey is mimicry (O’Shea, 2005). Scale pattern and coloration provide distinct boundaries in the wild and often carry unspoken warnings based on a reputation for snake venom toxicity for a given species. If a relatively harmless species is able to mimic the physical characteristics, especially color patterns, of a well-known highly toxic counterpart, then other potential predators may recognize both snakes as toxic and not pursue either.

Fang anatomy and dentition are good indicators of such factors as snake habitat and feeding habits (O’Shea, 2005). In general, the anatomical structure of fangs makes it nearly impossible for snakes to chew their prey. The distinct curvature of the fangs is engineered not only for puncturing skin and delivering venom but also for swallowing whole prey as well. The teeth and jaw structure are relatively mobile and effectively facilitate the positioning of whole prey for swallowing. The skull and jaw possess an extremely high degree of responsiveness to stimuli. The jaw does not actually dislocate; however, it is able to rapidly reposition itself to capture, contain, and swallow prey. In addition to the capabilities of the teeth, fangs, and jaws, the classic fork-shaped tongue is another tool for identifying prey (O’Shea, 2005).

The Viperidae fang structure is regarded as the most developed and efficient means of venom, or toxin, delivery to prey. The venom gland is positioned at the base of a long (~30 mm) hollow retractable fang (Mebs, 2002; Weinstein et al., 2010). Muscle pressure on the gland determines the amount of venom released. Another highly developed venom delivery apparatus is characteristic of the spitting cobras, aptly named for their ability to project venom via glands that protrude from the base of the fang opening (Mebs, 2002). Venom is carried toward the prey, or target, via forceful exhalation that is accompanied by a hissing sound. Toxin delivery via venom exposure is the primary mechanism by which snakes immobilize and kill their prey. Toxin type and specificity is dependent on the species; however, most venom consists of complex networks of toxins that affect variable organ systems and interact with one another increasing the overall potency.

It is estimated that there are over 2.5 million snakebites annually, and that over 100,000 victims will die (Mackessy, 2010; White, 2005). Information resources available to physicians on management of snakebite victims may be found at the Clinical Toxinology Resources Web site—www.toxinology.com—or other appropriate references (Dart, 2004; Tintinalli et al., 2004).

Snake Venoms

These venoms are complex mixtures: proteins and peptides, consisting of both enzymatic and nonenzymatic compounds, make up over 90% of the dry weight of the venom (Phui Yee et al., 2004). Snake venoms also contain inorganic cations such as sodium, calcium, potassium, magnesium, and small amounts of zinc, iron, cobalt, manganese, and nickel. The metals in snake venoms are likely catalysts for metal-based enzymatic reactions. For example, in the case of some elapid venoms, zinc ions appear to be necessary for anticholinesterase activity, and calcium may play a role in the activation of phospholipase A and the direct lytic factor. Some proteases appear to be metalloproteins. Some snake venoms also contain carbohydrates (glycoproteins), lipids, and biogenic amines, such as histamine, serotonin, and neurotransmitters (catecholamines and acetylcholine) in addition to positively charged metal ions (Russell, 2001; Mebs, 2002; Menez, 2003; Ramos and Selistre-de-Araujo, 2006). The complexity of snake venom components is illustrated nicely in Fig. 26-29 (Ramos and Selistre-de-Araujo, 2006).

Actions of snake venoms can be said to be broad ranging in several areas (O’Shea, 2005). A simplistic approach would group toxin components as neurotoxins, coagulants, hemorrhagins, hemolytics, myotoxins, cytotoxins, and nephrotoxins. Neurotoxins produce neuromuscular paralysis ranging from dizziness to ptosis; to ophthalmoplegia, flaccid facial muscle paralysis, and inability to swallow; to paralysis of larger muscle groups; and finally to paralysis of respiratory muscles and death by asphyxiation. Coagulants may have initial procoagulant action that uses up clotting factors leading to bleeding. Coagulants may directly inhibit normal clotting at several places in the clotting cascade or via inhibition of platelet aggregation. In addition, some venom components may damage the endothelial lining of blood vessels leading to hemorrhage. Bite victims may show bleeding from nose or gums, from the bite site, and in saliva, urine, and stools. Myotoxins can directly impact muscle contraction leading to paralysis or cause rhabdomyolysis or the breakdown of skeletal muscle. Myoglobinuria, or a dark brown urine, and hyperkalemia may be noted. Cytotoxic agents have proteolytic or necrotic properties leading to the breakdown of tissue. Typical signs include massive swelling, pain, discoloration, blistering, bruising, and wound weeping. Sarafotoxins, which are found only in burrowing asps of Afro-Arabia, cause coronary artery constriction that can lead to reduced coronary blood flow, angina, and myocardial infarction. Finally, nephrotoxins can cause direct damage to kidney structures leading to bleeding, damage to several parts of the nephron, tissue oxygen deprivation, and renal failure.

Enzymes

At least 26 different enzymes have been isolated from snake venoms, which can be a sequence of 150 to 1500 amino acids (Menez, 2003). No single snake venom contains all 26 enzymes and some important snake venom enzymes are shown in Fig. 26-29.

Proteolytic enzymes that catalyze the breakdown of tissue proteins and peptides include peptide hydrolases, proteases, endopeptidases, peptidases, and proteinases. Several proteolytic enzymes may be in a single venom. The proteolytic enzymes have molecular weights between 20,000 and 95,000. Some are inactivated by ethylenediaminetetraacetic acid (EDTA) and certain reducing agents. Metals appear to be intrinsically involved in the activity of certain venom proteases and phospholipases. The crotalid venoms examined so far appear to be rich in proteolytic enzyme activity. Viperid venoms have lesser amounts, whereas elapid and sea snake venoms have minimal, if any, proteolytic activity. Venoms that are rich in proteinase activity are associated with marked tissue destruction (Russell, 2001).

Collagenase is a specific kind of proteinase that digests collagen. This activity has been demonstrated in the venoms of a number of species of crotalids and viperids. The venom of Crotalus atrox (Fig. 26-30) digests mesenteric collagen fibers but no other proteins. EDTA inhibits the collagenolytic effect, but not the argine esterase effect.

Hyaluronidase cleaves internal glycoside bonds in certain acid mucopolysaccharides resulting in a decrease in the viscosity of connective tissues. The breakdown in the hyaluronic barrier allows other fractions of venom to penetrate the tissues, causing hyaluronidase to be called “spreading factor.” The degree to which hyaluronidase contributes to the extent of edema produced by the whole venom is not known (Kemparaju et al., 2010).

Phospholipase A2 (PLA₂) enzymes are widely distributed throughout the tissues of animals, plants, and bacteria, and have been well studied in snake venoms (Doley et al., 2010; Ohno et al., 2003; Soares and Giglio, 2003). Mammalian PLA₂ enzymes are involved in fertilization and cell proliferation, and have been implicated in respiratory ailments such as asthma, as well as skin conditions such as psoriasis (Kini, 2003; Soares and Giglio, 2003). PLA₂ catalyzes the Ca²⁺-dependent hydrolysis of the 2-acyl ester bond, producing free fatty acids and lysophospholipids. However, liberation of pharmacologically active products and effects independent of enzymatic action may contribute to their overall action. In general, mammalian PLA₂s are considered to be nontoxic and do not elicit the same pharmacologic effects as similar enzymes in snake venoms.

As of 2003, over 280 PLA₂ enzymes had been classified, sharing at least 40% identity in their amino acid sequences (Kini, 2003; Soares and Giglio, 2003). Many PLA₂s have been sequenced. They have approximately 120 amino acids and 14 cysteine residues forming seven disulfide bonds. Surface residue recognition and covalent/noncovalent bonds stabilize complexes for receptor binding. Specific amino acid residues play an important role in the diversification and overall function of the PLA₂s. Of note, histidine, lysine, cysteine, and methionine have been well studied for their contribution to enzyme structure and function (Soares and Giglio, 2003). For example, alkylation of His48 diminishes the hydrolytic capabilities of certain toxins. Alkylation of amino acids has proven to effectively mitigate PLA₂ toxicity and has been proposed as an antivenom treatment (Soares and Giglio, 2003). PLA₂s interact with other toxins in the venom as well, often resulting in synergistic reactions. Similarly, snake venom PLA₂ enzymes can be separated into three major groupings depending on their pharmacologic activities: low-toxicity enzymes (LD₅₀ >1 mg/kg), high-toxicity enzymes (1 mg/kg > LD₅₀ > 0.1 mg/kg), and presynaptically acting toxins (LD₅₀ <0.1 mg/kg) (Rosenberg, 1990). Although the sequences of these enzymes are homologous and their enzymatically active sites are identical, they differ widely in their pharmacologic properties. For example, taipoxin, a PLA₂ enzyme from the venom of the Australian elapid Oxyuranus scutellatus (Fig. 26-31), has an intravenous LD₅₀ in mice of 2 μg/kg, whereas the neutral PLA₂ from Naja nigricollis (Fig. 26-32) has an LD₅₀ of 10,200 μg/kg, even though N. nigricollis PLA₂ is enzymatically more active (Russell, 2001).

Arginine ester hydrolase is one of a number of noncholinesterases found in snake venoms. The substrate specificities are directed to the hydrolysis of the ester or peptide linkage, to which an argine residue contributes the carboxyl group. This activity is found in many crotalid and viperid venoms and some sea snake venoms but is lacking in elapid venoms with the possible exception of Ophiophagus hannah. Some crotalid venoms contain at least three chromatographically separable arginine ester hydrolases. The bradykinin-releasing and perhaps bradykinin-clotting activities of some crotalid venoms may be related to esterase activity.

Two distinct classes of fibrin(ogen)olytic enzymes, the metalloproteinases and the serine proteinases, have been isolated from venom of Viperidae, Elapidae, and Crotalidae snake families (Swenson and Markland, 2005). These two classes of proteinases differ in mechanism of action and their target in fibrin(ogen), but ultimately they break down fibrin-rich clots and help to prevent further clot formation. The properties of fibrolase, an α-chain fibrinolytic metalloproteinase from Agkistrodon contortrix contortrix (Fig. 26-33) venom, and β-fibrinogenase, a β-chain fibrinogenase from Vipera lebetina, are provided in Table 26-13. Properties of some additionally characterized fibrin(ogen)ases are listed in Table 26-14. It is apparent that there are major differences in the properties of these enzymes from different snakes even though they have similar catalytic properties. An exciting development from the research on these enzymes is that one specific recombinant fibrinolytic enzyme derived from fibrolase called alfimeprase is progressing through clinical trials for the treatment of peripheral arterial occlusions.

The snake venom hemorrhagic metalloproteinases (SVMP) are enzymes that disrupt the hemostatic system and they are characterized by their domain structure into four primary classes, PI to PIV (Fox and Serrano, 2010). SVMPs are synthesized in vivo as multimodular proteins that comprise a signal peptide, a prodomain, and a metalloprotease domain (SVMP PI). SVMP potency tends to increase by class and those within the PIV class are larger and comprise additional disulfide bonds (Calvette et al., 2003, 2005). Class PII proteins exhibit a C-terminal disintegrin domain. The PII metalloproteases block the function of integrin receptors, a function that could alleviate a variety of pathological conditions such as inflammation, tumor angiogenesis and metastasis, and thrombosis. The integrin-blocking specificity of this class of metalloproteins is highly dependent on the conformation of the inhibitory loop, and thus the placement and bonding of cysteine residues. More specifically, within the inhibitory loops, RGD-containing disintegrins are specific to the class PII metalloproteases (Calvette et al., 2003, 2005). Class PIII SVMPs exhibit the disintegrin-like domain and the C-type lectin-like domain is present in PIV SVMPs. The metalloproteinase domain or catalytic domain is composed of about 215 amino acids and has metal-dependent endopeptidase activity (Calvette et al., 2005). SVMPs degrade proteins such as laminin, fibronectin, type IV collagen, and proteoglycans from the endothelial basal membrane; degrade fibrinogen and von Willebrand factor enhancing the hemorrhagic action; and inhibit platelet aggregation and stimulate release of cytokines (Ramos and Selistre-de-Araujo, 2006).

The proteolytic action of thrombin and thrombin-like snake venom enzymes is shown in Table 26-15. This table compares ancrod (from Calloselasma rhodostoma), batroxobin (from Bothrops moojeni), crotalase (from Crotalus adamanteus), gabonase (from Bitis gabonica), and venzyme (from A. contortrix). Table 26-16 shows the molecular size of some thrombin-like enzymes. A recent contribution on snake toxins, using mass spectrometric immunoassay and bioactive probe techniques, has been published by Ramirez et al. (1999a,b). Considerable study has been given to the hemostatic properties of venoms (Markland, 1998; Phillips et al., 2010). The hemostatically active components are summarized in Table 26-17.

Phosphomonoesterase (phosphatase) is widely distributed in the venoms of all families of snakes except the colubrids (Dhananjaya et al., 2010). It has the properties of an orthophosphoric monoester phosphohydrolase. There are two nonspecific phosphomonoesterases, and they have optimal pH at 5.0 and 8.5. Many types of venom contain both acid and alkaline phosphatases, whereas others contain one or the other.

Phosphodiesterase has been found in the venoms of all families of poisonous snakes. It is an orthophosphoric diester phosphohydrolase that releases 5-mononucleotide from the polynucleotide chain and thus acts as an exonucleotidase, attacking DNA and RNA. More recently, it has been found that it also attacks derivatives of arabinose.

Acetylcholinesterase was first demonstrated in cobra venom and is widely distributed throughout the elapid venoms (Ahmed et al., 2010). It is also found in sea snake venoms but is totally lacking in viperid and crotalid venoms. It catalyzes the hydrolysis of acetylcholine to choline and acetic acid. The role of the enzyme in snake venoms is not clear, but may function to catalyze the hydrolysis of acetylcholine thereby facilitating tetanic paralysis and capture of prey.

RNase is present in some snake venoms in small amounts as the endopolynucleotidase RNase. It appears to have specificity toward pyrimidine-containing pyrimidyladenyl bonds in DNA. The optimum pH is 7 to 9 when ribosomal RNA is used as the substrate. This enzyme in Naja oxiana venom has a molecular weight of 15,900.

DNase acts on DNA to produce predominantly trinucleotides or higher oligonucleotides that terminate in 3′-monoesterified phosphate. C. adamanteus venom contains two DNases, with optimum pH at 5 and 9.

5′-Nucleotidase is a common constituent of all snake venoms; in most instances it is the most active phosphatase in snake venoms. It specifically hydrolyzes phosphate monoesters, which link with a 5′ position of DNA and RNA. It is found in greater amounts in crotalid and viperid venoms than in elapid venoms. The molecular weight as determined from amino acid composition and gel filtration with Naja naja atra venom has been estimated at 10,000. The enzyme from N. naja venom is enhanced by Mg²⁺, is inhibited by Zn²⁺, is inactivated at 75°C at pH 7.0 or 8.4, and has an isoelectric point of about 8.6. That from Agkistrodon halys blomhoffi shows a pH optimum of 6.8 to 6.9, with activity being enhanced by Mg²⁺ and Mn²⁺ and inhibited by Zn²⁺. The enzyme has a low order of lethality (Russell, 2001).

Nicotinamide adenine dinucleotide (NAD) nucleotidase has been found in a number of snake venoms. This enzyme catalyzes the hydrolysis of the nicotinamide N-ribosidic linkage of NAD, yielding nicotinamide and adenosine diphosphate riboside. Its optimum pH is 6.5 to 8.5; it is heat labile, losing activity at 60°C. Nucleotidases function as ADP scavengers thereby acting as potent inhibitors of platelet aggregation.

l-Amino acid oxidase has been found in all snake venoms examined so far and it is what gives the characteristic yellow color to the venom. This enzyme catalyzes the oxidation of l-α-amino and α-hydroxy acids. This activity results from a group of homologous enzymes with molecular weights ranging from 85,000 to 150,000. The mouse intravenous LD₅₀ of the enzyme from C. adamanteus venom was 9.13 mg/kg body weight, approximately four times less than the lethal value of the crude venom, and this enzyme had no effect on nerve, muscle, or neuromuscular transmission (Russell, 2001; Tan and Fung, 2010).

Polypeptides

Snake venom polypeptides are low-molecular-weight proteins that do not have enzymatic activity. More than 80 polypeptides with pharmacologic activity have been isolated from snake venoms. Interested readers will find definitive reviews on these peptides in the works of Lee (1979), Eaker and Wadström (1980), and Gopalakrishnakone and Tan (1992). Most of the lethal activity of the poison of the sea snake Laticauda semifasciata was recovered as two toxins, erabutoxin-a and erabutoxin-b, using carboxymethylcellulose chromatography; 30% of the proteins were erabutoxins. More recently, erabutoxin-a, a short-chain curamimetic, has been crystallized in monomeric and dimeric forms (Nastopoulos et al., 1998). Erabutoxin-b is said to be relatively ineffective at the mammalian neuromuscular junction (Vincent et al., 1998). Another curamimetic, a long-chain polypeptide, is α-cobratoxin, while a novel “neurotoxin” from N. naja atra, having 61 amino acid residues and eight cystine residues, has been isolated by Chang et al. (1997).

Disintegrins are a family of short cysteine-rich polypeptides and are divided into five subgroups based on the combination of length and number of disulfide bonds of polypeptides. In general, the disintegrins comprise 40 to 100 amino acids. Their small size coupled with a relatively dense network of disulfide bonds contributes to the tertiary structure of these compounds and high potency of such small compounds. Disintegrins are released in venoms via proteolytic processes of PII metalloproteinases, whereas structures similar in form and function to disintegrins, or disintegrin-like, are subject to PIII processes (Calvette et al., 2005). Monomeric disintegrins can vary from about 50 residues and four disulfide bonds as in echistatin and obtustatin to around 70 amino acid residues and six disulfide bridges as in albolabrin, barbourin, and halysin, to over 84 amino acids and seven disulfide bonds for bitistatin and salmosin-3. Dimeric disintegrins are about 67 amino acids long and contain four intrachain disulfide linkages and two between-chain bonds. Examples include contortrostatin and acostatin. The monomeric disintegrin-like chemicals contain around 100 amino acids and eight disulfide bonds, and include trimelysin-I, bothropasin, and jararhagin (Calvette et al., 2003; Ramos and Selistre-de-Araujo, 2006).

RGD (argine–glycine–asparagine) and non-RGD-containing disintegrins coexist in certain venoms and exhibit affinities for variable ligand receptors. In such cases, one copy of the gene encodes for the more conserved RGD function of platelet aggregation, whereas the duplicated genes have drifted toward facilitating other biological functions. Modeling and structure analysis of cyclic RGD peptides has implicated the importance of amino acid sequences on the C-terminal of the RGD sequence; furthermore, the physical features such as size of the integrin-binding loop contribute to the receptor-binding capabilities (Calvette et al., 2005). In general, the amino acid residues of this region of the RGD sequence are not well conserved and are thought to play a key role in determining integrin receptor-binding specificity. There are additional mechanisms within the C-terminal region, which include conformational epitopes that are utilized to alter receptor-binding capabilities.

The small basic polypeptide myotoxins are widely distributed in Crotalus snake venoms. The specific agent crotamine from Crotalus durissus terrificus venom induces skeletal muscle spasms and paralysis by changing the inactivation process of sodium channels, which are inhibited by tetrodotoxin and potentiated by veratridine and grayanotoxin, leading to depolarization of the neuromuscular junction. Crotamine is composed of 42 amino acid residues and three disulfide bonds. The crotamine gene contains 1.8 kbp and has three exons that are separated by a long phase-1 and a short phase-2 intron and mapped to chromosome 2. In addition, the three-dimensional structure has been published, and the structural topology is similar to that of other three disulfide bridge containing peptides such as human β-defensins and scorpion sodium channel toxin. These structural properties enable crotamine to have a unique cell penetrating ability allowing the toxin to concentrate in the nucleus by means of a probable receptor-independent mechanism. It is interesting to note that topology and diversification of functional folds are common themes in animal venom peptides acting on ion channels and other targets (Menez, 1998; Mouhat et al., 2004; Oguiura et al., 2006).

Toxicology

In general, the venoms of rattlesnakes and other New World crotalids produce alterations in the resistances and often in the integrity of blood vessels, changes in blood cells and blood coagulation mechanisms, direct or indirect changes in cardiac and pulmonary dynamics, and—with crotalids such as C. durrissus terrificus and C. scutulatus—serious alterations in the nervous system and changes in respiration. In humans, the course of the poisoning is determined by the kind and amount of venom injected; the site where it is deposited; the general health, size, and age of the patient; the kind of treatment; and those pharmacodynamic principles noted earlier in this chapter. Death in humans may occur within less than one hour or after several days, with most deaths occurring between 18 and 32 hours. Hypotension or shock is the major therapeutic problem in North American crotalid bites (Russell, 2001).

Snakebite Treatment

The treatment of bites by venomous snakes is now so highly specialized that almost every envenomation requires specific recommendations. However, three general principles for every bite should be kept in mind: (1) snake venom poisoning is a medical emergency requiring immediate attention and the exercise of considerable judgment; (2) the venom is a complex mixture of substances of which the proteins contribute the major deleterious properties, and the only adequate antidote is the use of specific or polyspecific antivenom; and (3) not every bite by a venomous snake ends in an envenomation. Venom may not be injected. In almost 1000 cases of crotalid bites, 24% did not end in a poisoning. The incidence with the bites of cobras and perhaps other elapids is probably higher. The reader is referred to other appropriate texts for appropriate treatment of snakebites (Russell, 2001; Dart, 2004; Sholl et al., 2004; Tintinalli et al., 2004; Singletary and Holstege, 2006; Smith and Bush, 2010; see also www.toxinology.com).

Snake Venom Evolution

Considerable efforts are being expended to examine the complex process by which snake venom components are thought to have changed over the years. This evaluation involves tracing the ancestral roots of toxins, which is made even more cumbersome due to the distinct differences in the speed at which individual components of a venom evolve. The current assemblage of snake venoms with regard to functionality and ancestral protein activity is outlined in Table 26-18 (Fry, 2005). In general, the toxins from ancestral proteins that were constructed of dense networks of cysteine cross-linkages are considered among the most diverse today in terms of toxicological insult.

The evolution of the PLA₂s appears to be directed toward modifying the molecular surface in order to maximize the diversity of the pharmacologic properties of each isozyme component (Kini and Chan, 1999; Li et al., 2005). As PLA₂s are not crucial for snake survival, mutations in some PLA₂ isozymes may be ignored and allowed to progress, thereby expediting the evolution in comparison to other more critical enzymes in which mutations are monitored more closely and not allowed to progress. The functionality of PLA₂ isozymes can be significantly hindered by increased rates of mutations within highly conserved regions of the amino acid sequence, which could in turn displace cysteine residues and alter the tertiary protein structure. However, increased mutation of a particular subset of enzymes, such as the PLA₂s, may be a result of a shift in ecological niche. A recent study (Li et al., 2005) reports a shift in the diet composition of the marbled sea snake as the root cause of decelerated evolution of the PLA₂ enzymes. The diet change from live prey to fish eggs essentially negated the need for PLA₂ enzymes to immobilize live prey for swallowing and digestion. Future research could focus on the declining need for PLA₂s, and on the altered digestive enzyme composition that has likely occurred as a result of diet shift.

Antivenoms have been produced against most medically important snake, spider, scorpion, and marine toxins. Animals immunized with venom develop a variety of antibodies to the many antigens in the venom. Antivenom consists of venom-specific antisera or antibodies concentrated from immune serum to the venom. Antisera contain neutralizing antibodies: one antigen (monospecific) or several antigens (polyspecific). Monovalent antivenoms have a high neutralization capacity, which is desirable against the venom of a specific animal. Polyvalent antisera are typically used to cover several venoms, such as snakes from a geographic region. Polyvalent preparations usually required higher doses or volumes than monovalent antivenoms. Neutralization capacity of antivenom is highly variable as there are no enforced international standards. Antivenom may cross-react with venoms from distantly related species and may not react with venom from the intended species. Nevertheless, in general, the antibodies bind to the venom molecules, rendering them ineffective.

Antivenoms are available in several forms: intact IgG antibodies or fragments of IgG such as F(ab)₂ and Fab. The molecular weight of the intact IgG is about 150,000, whereas that of Fab is approximately 50,000. The molecular size of IgG prevents its renal excretion and produces a volume of distribution much smaller than that of Fab. The elimination half-life of IgG in the blood is approximately 50 hours. Its ultimate fate is not known, but most IgG is probably taken up by the reticuloendothelial system and degraded with the antigen attached. Fab fragments have an elimination half-life of about 17 hours, and are small enough to permit renal excretion.

All antivenom products may produce hypersensitivity reactions. Type I (immediate) hypersensitivity reactions are caused by antigen cross-linking of endogenous IgE bound to mast cells and basophils. Binding of antigen by a mast cell may cause the release of histamine and other mediators, producing an anaphylactic reaction. Once initiated, anaphylaxis may continue despite discontinuation of antivenom administration. Type III hypersensitivity (serum sickness) may develop several days after antivenom administration. In these cases, antigen–antibody complexes are deposited in different areas of the body, often producing inflammatory responses in the skin, joints, kidneys, and other tissues. Fortunately, these reactions are rarely serious. The risks of anaphylaxis should always be considered when one is deciding whether to administer antivenom, and thus antivenom should be given only by intravenous infusion under medical supervision (Heard et al., 1999; Russell, 2001; Mebs, 2002; Dart, 2004; Tintinalli et al., 2004; www.toxinology.com).

Animal venoms are being used as research and clinical tools based on their high affinity for specific targets and well-studied pharmacologic properties (Cherniack, 2010, 2011; Mayer et al., 2010; Menez, 1998, 2003; Dimarcq and Hunneyball, 2003; Escoubas and Rash, 2004). Toxin specificities for receptors and channels that facilitate the interface and coordination of neuromuscular activity are utilized and manipulated to study, model, diagnose, and sometimes treat acute and degenerative conditions. On closer examination of α-bungarotoxin and candoxin nicotinic acetylcholine receptor specificity, plans are under way to utilize the reversible and irreversible receptor binding in muscular and neuronal tissues, respectively, in Alzheimer patients (Phui Yee et al., 2004). In addition to treating neurological diseases, specific α-toxins (longer chained) are also studied for their antiangiogenic capabilities in treating malignant tumor growth in patients suffering from small cell lung carcinoma (Tsetlin and Hucho, 2004). In cases such as this, there is an inherent trade-off between promoting some degree of neurological deficit in light of combating tumor growth. Toxins such as the snake venom thrombin-like enzymes are valuable tools in both research and therapeutic applications. Similarly, fibrin(ogen)olytic enzymes that break down fibrin-rich clots preventing further clot formation may be useful as controls in blood clotting research or to treat heart attacks and strokes (Castro et al., 2004; Marsh and Williams, 2005; Swenson and Markland, 2005). To facilitate research, the complement-activating cobra venom factor has been produced by recombinant techniques (Vogel et al., 2004). These pharmazooticals include reptilase-R, bothrocethin, stypven, ecarin, and protac.

Active research efforts have noted that animal venoms contain components that can reduce pain, can selectively kill specific cancers, may reduce the incidence of stroke via effects on blood coagulability, and function as antibiotics. Numerous compounds have been isolated from marine animals (Bingham et al., 2010; Newman et al., 2009), including the anticancer agents plinabulin, tasidotin, bryostatin 1, hemiasterlin, and salinosporamide A (also known as marizomib). The compound epibatidin comes from the skin of the South American frog, Epipedobates tricolor, and a synthetic derivative ABT-594 appears to be more effective than morphine without being addictive. TM 601 is derived from the Israeli yellow scorpion and attacks malignant brain tumors called glioma tumors responsible for two-thirds of the cases of brain cancer, without harming healthy cells. ET 743, which comes from sea squirts, is being tested for treatment of ovarian cancer and soft tissue sarcoma. Ancrod is an anticoagulant with potential to prevent cell damage and death when someone suffers a stroke. The active ingredient comes from the venom of the Malaysian pit viper. In Germany, where Ancrod has been marketed for a number of years, a specially built facility houses about 3000 snakes. Several other sources of anticoagulants are being examined. A substance called magainin 2, which comes from the skin of frogs, is an effective antibiotic to which bacteria do not appear to develop resistance. The clotting enzyme batroxobin is an ingredient in reptilase and has been used in the development of fibrin glue, which is used in surgery to stop diffuse bleeding from liver or lung by covering the surface with a thin layer of fibrin.

Another major area of investigation and success involves the venom components that act as enzyme inhibitors. In particular, venom peptides from Bothrops jararaca were initially called bradykinin-potentiating peptides and lowered blood pressure. After further research, it became clear that these peptides were inhibitors of angiotensin I–converting enzyme, and chemical modification leads to orally active agents such as captopril.

Venom toxins can also be used as a component of the toxin–receptor–antibody complex for diagnosis of autoimmune disorders (Menez, 2003). In addition to providing a promising means for researching and treating muscular and neurological diseases and cancer, work is being conducted on designing methods by which to consistently reconstruct and conform the overall toxin structure to bind to a specific protein, such as HIV (Menez, 2003).

It has been shown that leeches, earthworms, helminths, snails, centipedes, spiders, and ticks all produce substances with potential clinical applications, such as osteoarthritis, deep vein thrombosis, antimicrobial action, inflammatory bowel disease, analgesia, and hyperlipidemia (Cherniack, 2011). In particular, approximately 14 anticoagulant chemicals have been isolated from the leech, including hirudin and lepirudin, apyrase, collagenase, hyaluronidase, and eglin (Lubenow and Greinacher, 2002; Massart et al., 2009). Tick saliva contains proteins that inhibit human kallikrein and factors XIa and XIIa (Mazzuca et al., 2007). Earthworms contain proteins with anticoagulant, antimicrobial, and anti-inflammatory bowel disease properties (Joo et al., 2009; Reddy and Fried, 2009; Ruyssers et al., 2010). Bees can provide honey, royal jelly, and propolis, which all have antimicrobial properties, and the venom contains apamin and mellitin, which have anti-inflammatory properties. Additional details of the clinical applications of animal saliva and venom may be found in Cherniack (2010, 2011).

Additional work is being conducted on animals such as the mongoose, hedgehog, and opossum, which all embody a high level of resistance to snakebites. Blood from these animals contains proteins between 400 and 700 amino acids long that inhibit hemorrhagins. The exact mechanism of the many components in animal venoms that produce toxicity or resistance to certain toxins has yet to be determined. Further research will require a multidisciplinary approach involving techniques from parasitology, chemistry, molecular biology, genomics, proteomics, physiology, pharmacology, and toxicology.

The myriad toxins produced by plants and animals range from the relative simplicity of small chemical agents to the exceedingly complex proteinaceous toxins. The effects of these compounds are amazingly diverse and can range from local irritation to systemic destruction and death. The interplay between toxin and organism is often hard to study due to difficulty involved in recreating the interaction in the laboratory. One must also not forget the interactions that arise between toxins and substances already present in the organism. These interactions can act to worsen or reduce the poisonous outcome. As laboratory techniques become more sophisticated and new methods are developed, research concerning toxins and their effects will continue to grow.

The author gratefully acknowledges the assistance of Peter Joseph Massa in the preparation of this chapter.