Botulinum Toxin: From Poison to Medicinal Agent
by Jiri Patocka^a and Miroslav Splino^b

^a Department of Toxicology, Military Medical Academy, Hradec Kralove and Faculty of Health and Social Care, University of South Bohemia, Ceské Budejovice,
^b Department of Epidemiology, Military Medical Academy, Hradec Kralove, Czech Republic

Introduction:
          Botulinum toxin is very strong poison produced by the microorganism Clostridium botulinum. C. botulinum is classified as a single species but consists of at least three genetically distinguishable groups of organisms. These are alike in their abilities to produce neurotoxins with similar pharmacological activities [1] but diverse serologic properties (toxin types A, B, C, D, E, F, and G). These types are defined by the International Standards for Clostridium botulinum Antitoxin [2]. Botulinum toxins are the causative agents of botulism, a potentially fatal condition of neuromuscular paralysis. Botulism is characterized by symmetric, descending, flaccid paralysis of motor and autonomic nerves, usually beginning with the cranial nerves. Blurred vision, dysphagia, and dysarthria are common initial complaints. The diagnosis of botulism is based on compatible clinical findings; history of exposure to suspect foods; and supportive ancillary testing to rule out other causes of neurological dysfunction that mimic botulism, such as stroke, Guillain-Barré syndrome, and myasthenia gravis. Treatment includes supportive care and trivalent equine antitoxin, which reduces mortality if administered early.
          Clinicians are the first to treat patients in any type of botulism outbreak. It is important that they know how to recognize, diagnose, and treat this rare but potentially lethal disease. Recently, the potential terrorist use of botulinum toxin has become an important concern. Human botulism is primarily caused by the strains of C. botulinum that produce toxin types A, B, and E. The strains of C. baratii [3], which produce type F toxin and C. butyricum [4], which produces type E toxin, also have been implicated in human botulism. Strains of C. botulinum that produce type C or type D toxin for the most part cause botulism only in nonhuman species. All these neurotoxigenic organisms are anaerobic, gram-positive, spore-forming bacilli and are commonly found in soils throughout the world. C. botulinum organisms cause food poisoning because the heat-resistant spores survive food preservation methods that kill non-sporulating organisms; they subsequently produce a potent neurotoxin under anaerobic, low-acid (pH > 4.6), and low solute conditions [5]. The toxins affect a broad range of vertebrate species, but the evolutionary utility of toxin production to the bacterial host organisms is unclear.

Epidemiology:
          Four clinical forms of botulism occur in humans: foodborne botulism; wound botulism; infant botulism; and, rarely, adult infectious botulism. Foodborne botulism is a public health emergency because the contaminated food may still be available to other people besides the patient. Studies in monkeys indicate that, if aerosolized, botulinum toxin also can be absorbed through the lungs [6]. The persistence of botulinum toxin is very high: it remains in nonmoving water and food for weeks. Important changes in the epidemiology of botulism have emerged in the past few decades. Recently identified vehicles for foodborne botulism include homemade salsa and traditionally prepared salted or fermented fish. In recent years, restaurant-associated outbreaks accounted for a large proportion of botulism cases. Botulism is not spread from one person to another. Foodborne botulism can occur in all age groups. Infant botulism occurs in a small number of susceptible infants each year who harbor C. botulinum in their intestinal tract. It usually affects children in the first year of life (mortality 2 %), with the onset of obstipation, lethargy, ptosis, swallowing discomfort, hypotonia, general weakness, and distressed breathing. It accounts for 5 - 10 % of the Sudden Infant Death Syndrome (SIDS).

The Structure of Botulinum Toxin and Mechanism of Toxic Action:
          All botulinum toxins are synthesized as single-chain polypeptides with a molecular Wight of approximately 150 kDa [7]. The complete amino acid sequences for the various serotypes are known [8] and regions of sequence homology among the serotypes suggest that all employ similar mechanism of biological action [9]. In the single-chain form, toxins have relatively little potency as neurotoxins. Neurotoxic activation requires a two-step modification in the tertiary structure of the protein [10]. In the first step, the parent chain is cleaved between amino acids 448 and 449. The result is one light chain (amino acids 1-448, approx. 100 kDa) and one heavy chain (amino acids 449-1295, approx. 100 kDa) connected via a disulfide bond. The light chain is associated with one atom of zinc [11] (Fig. 1). In this form, the toxin enters the axon terminal. The second activating step, disulfide reduction, occurs only after internalization by the target cell.
          Botulinum neurotoxins are potent blockers of synaptic transmission in peripheral cholinergic nervous system synapses, thereby causing paralysis [12]. Recent studies on the biochemical dissection of the components involved in the fusion of secretory vesicles with the plasma membrane have set botulinum toxins at the center of this process. Botulinum toxins are Zn2+-metalloproteases that selectively cleave proteins implicated in the fusion process and, accordingly, block neurotransmitter release into the synaptic cleft [13-15]. Botulinum toxin A, E, and C proteolyse the plasma membrane associated proteins SNAP-25 (synaptosome associated protein of 25 kDa). Botulinum toxins A and E cleave SNAP-25 and botulinum toxin C cleave still syntaxin [16-17]. Botulinum toxins B, D, F, and G proteolyze synaptobrevin, a vesicle-associated membrane protein, also known as VAMP [14]. The protease activity of the toxins is confined to their light chains [15]. Botulinum toxins have an inherent propensity to insert into membranes, especially at acidic pH. This property is compatible with the ion-channel activity observed when Botulinum toxins B, C, D, and E are reconstituted in lipid bilayers [18]. A novel combination of theoretical approaches was exploited to predict which amino acid residues of various botulinum neurotoxin serotypes participate in forming ion channels [19]. Image reconstruction analysis of electron micrographs of botulinum toxin inserted in membranes suggests the occurrence of a tetramer as the structural entity underlying the botulinum toxin channel [20].

Clinical Features of Botulism:
          Foodborne botulism is caused by ingestion of preformed toxin produced in food by C. botulinum. The most frequent source is home-canned foods, in which spores that survive an inadequate cooking and canning process germinate and produce toxin in the anaerobic environment of the canned food. In the event of intentional foodborne poisoning with botulinum toxin, the signs and symptoms developing after ingestion would probably resemble those of naturally occurring foodborne botulism. If aerosolized toxin was inhaled, the incubation period might be slightly longer and gastrointestinal symptoms might not occur [6]. The clinical syndrome of foodborne botulism is dominated by neurologic symptoms resulting (cont.p.16 - Botox) (Botox - from p. 15) from a toxin-induced block of the voluntary motor and autonomic cholinergic junctions. With foodborne botulism, symptoms begin within 6 hours to 2 weeks (most commonly between 12 and 36 hours) after eating toxin-containing food. Although the syndrome is similar for each toxin type, type A toxin has been associated with more severe disease and a higher fatality rate than type B or type E toxin [24]. Symptoms from any toxin type may range from subtle motor weakness or cranial nerve palsies to rapid respiratory arrest. The initial symptoms of foodborne botulism may be gastrointestinal and can include nausea, vomiting, abdominal cramps, or diarrhea; after the onset of neurologic symptoms, constipation is more typical. Dry mouth, blurred vision, and diplopia are usually the earliest neurologic symptoms. These initial symptoms may be followed by dysphonia, dysarthria, dysphagia, and peripheral muscle weakness. Symmetric descending paralysis is characteristic of botulism. Paralysis begins with the cranial nerves, the upper extremities, the respiratory muscles, and, finally, the lower extremities in a proximal-to-distal pattern. Onset usually occurs 18 to 36 hours after exposure [25]. In severe cases, extensive respiratory muscle paralysis leads to ventilatory failure and death unless supportive care is provided. Patients have required ventilatory support for up to 7 months before the return of muscular function, but ventilatory support is most commonly needed for 2 to 8 weeks. Clinical recovery generally occurs over weeks to months; electron microscopic evidence suggests that clinical recovery correlates with the formation of new presynaptic end plates and neuromuscular junctions [26]. Before mechanical ventilation and intensive supportive care, up to 60% of patients died. Death now occurs in 5% to 10% of cases of foodborne botulism; early deaths result from a failure to recognize the severity of disease, whereas deaths after 2 weeks result from complications of long-term mechanical ventilatory management [25]. Administration of trivalent or heptavalent botulinum antitoxin is recommended as the treatment for Botulinum toxin poisoning (in USA, CDC - Center for Disease Control, Atlanta, Georgia).

Military and Terrorist Misuse of Botulinum Toxin:
          There is a heightened awareness of the threat of biological weapons being used for biological warfare or bioterrorism. Many toxins that may be used as such biological weapons can easily be acquired and mass-produced. Dissemination of aerosols of these biological agents can produce mass casualties. If used by a terrorist they may overwhelm our current public health system. Some biological agents, such as Bacillus anthracis (anthrax) and botulinum toxin, are considered far more likely than others to be used as biological weapons. The potential for intentional poisoning with botulinum toxin has come into clearer focus in recent years [27]. As many as 17 countries were suspected to include or to be developing biological agents in their offensive weapons programs in 1996 [28] and at present the number of countries is probably even higher. Botulinum toxin often is included as one of these agents because it is relatively easy to produce and is highly lethal in small quantities. In August 1995, Iraq revealed that during the Gulf War, 11 200 L of botulinum toxin preparation was loaded into specially designed SCUD missile warheads [29]. In addition, before the Aum Shinrikyo used sarin in the 1995 terrorist attack on the Tokyo subway system, the cult had produced botulinum toxin [30].
          Development and use of botulinum toxin as a possible biological weapon began at least 60 years ago [31]. The head of the Japanese biological warfare group (Unit 731) admitted to feeding cultures of Clostridium botulinum to prisoners with lethal effect during that country's occupation of Manchuria, which began in the 1930s [32]. The US biological weapons program first produced botulinum toxin during World War II. Because of concerns that Germany had weaponized botulinum toxin, more than 1 million doses of botulinum toxoid vaccine were made for Allied troops preparing to invade Normandy on D-Day [33].
          Although the 1972 Biological and Toxin Weapons Convention prohibits offensive research and production of biological weapons, signatories Iraq and the Soviet Union subsequently produced botulinum toxin for use as a weapon [34]. Botulinum toxin was one of several agents tested at the Soviet site Aralsk-7 on Vozrozhdeniye Island in the Aral Sea [35]. A former senior scientist of the Russian civilian bioweapons program reported that the Soviets had attempted splicing the botulinum toxin gene from C. botulinum into other bacteria [36]. With the economic difficulties in Russia after the demise of the Soviet Union, some of the thousands of scientists formerly employed by its bioweapons program have been recruited by nations attempting to develop biological weapons [37]. Four of the countries listed by the US government as "state sponsors of terrorism" (Iran, Iraq, North Korea, and Syria) have developed, or are believed to be developing, botulinum toxin as a weapon [38, 39].
          After the 1991 Gulf War, Iraq admitted to the United Nations inspection team to having produced 19 000 L of concentrated botulinum toxin, of which approximately 10 000 L were loaded into military weapons [40]. These 19 000 L of concentrated toxin are not fully accounted for and constitute approximately 3 times the amount needed to kill the entire current human population by inhalation. In 1990, Iraq deployed specially designed missiles with a 600-km range; 13 of these were filled with botulinum toxin, 10 with aflatoxin, and 2 with anthrax spores. Iraq also deployed special 400-lb (180-kg) bombs for immediate use; 100 bombs contained botulinum toxin, 50 contained anthrax spores, and 7 contained aflatoxin [40]. It is noteworthy that Iraq chose to weaponize more botulinum toxin than any other of its known biological agents.
          Some contemporary analyses discount the potential of botulinum toxin as a toxin weapon because of constraints in concentrating and stabilizing the toxin for aerosol dissemination. But the terrorist use of botulinum toxin in the deliberate contamination of food could produce either a large botulism outbreak from a single meal or episodic, widely separated outbreaks [41]. In the US, the CDC maintains a well-established surveillance system for human botulism based on clinician reporting that would promptly detect such events [42].

Botulinum Toxin in Medicine:
          Viewed in a therapeutic context, two properties of botulinum toxin have excited a great deal of interest: 1) its ability to pass from the gut into general circulation has raised the possibility that it might act as a carrier for oral medications; and 2) its ability to bind with high affinity to cholinergic nerve endings and specifically block ACh release has suggested that it might be useful in treating disorders of cholinergic transmission. Preliminary studies of botulinum toxin as a carrier for oral medications look quite promising but have not yet come to fruition [43]. The use of botulinum toxin as a medicinal agent for correcting cholinergic disorders is very promising [44] and be used in spasmatic muscular disorders such as:

• spasmodic dysphonia which affects the larynx muscles and results in speech that is difficult to understand
• spasmodic torticollis, contractions of the neck and shoulder muscles
• blepharospasm, uncontrollable spasm of the eyelids
• oromandibular dystonia, clenching of the jaw muscles
• and the treatment of tics and cerebral palsy

          Botulinum toxin A has had a lot of publicity recently for its cosmetic use in facial wrinkles [45]. It is currently in widespread use for the treatment of glabellar frown lines, crow's feet, and horizontal forehead lines. Other more recent uses include extended management of cosmetic problems, including platysmal bands and horizontal neck lines as well as lines in the lower part of the face. Small doses of the toxin are injected into the affected muscles. As happens with botulism, the toxin binds to the nerve endings, blocking the release of the chemical acetylcholine, which would otherwise signal the muscle to contract. The toxin thus paralyzes or weakens the injected muscle but leaves the other muscles unaffected. This effect is temporary and it is possible to repeat it each about half year.
          A less visible, but socially important use of Botox is in the treatment of hyperhidrosis, which is excessive, sweating of the underarms, hands, and/or feet. Botox is injected at the site of the problem, underarms, foot or palm, and works by blocking the signal from the nerve to the sweat gland, thereby eliminating excessive moisture. Botox will not eliminate odor, the glands responsible for sweat and odor are different. The previous treatment for hyperhidrosis involved major surgery, actually cutting the nerves that caused sweating, thereby, disrupting the signal to the sweat glands.
          Botulinum toxin-treatment results are noticeable within two to four days after the treatment. Injections usually have to be repeated, as the effects are temporary and usually last about three to four months, although sometimes they can last over a year. This is not a cure for chronic diseases, but a definite improvement over the medical and surgical treatments previously available. Most complications occur when the toxin affects muscles other than those intended. For example, when being treated for crows feet, the toxin may diffuse into the eyelid muscle and cause a slight drooping of the eyelid, ptosis. This weakness usually resolves within a few days to a few weeks. Since botulinum toxin has not been in use for many years, long-term effects are unknown, but no lasting side effects have been reported. It is not recommended for use by pregnant or nursing women. Botulinum toxin should be used cautiously in persons with myasthenia gravis and Eaton-Lambert syndrome.

Editor's Note:

1. Botulinum toxins are very potent toxins with real potential as a biological agent for both warfare and terrorism. They also have great utility in medicine to treat spasmodic muscle disorders. Ironically, the more common they become in medical treatment, the more of a potential threat they become because they are produced commercially in relatively large amounts. But to put this in perspective, BotoxA is sold in vials of 100 U (20 U = 1 ng) as stated on the package insert. Even though it may only take 90 g to contaminate a typical large reservoir, it will take over a billion of these vials to contaminate the reservoir to 1 ng/L.
2. . The toxicity of botulinum toxin is usually expressed in mouse unit (U), where one unit (U) of Botulinum toxin A is the median intraperitoneal lethal dose (LD₅₀) in mice and is approximately 20 units/nanogram (1U=about 0.05 ng). The lethal dose in humans is not known, but extrapolating monkey data [21, 22] to a 70 kg human implies a parenteral lethal dose of nearly 3000 U, i.e., 4.3 µg [23]. According to JAMA, 285, 28 Feb 2001, "Botulinum Toxin as a Biological Weapon" by Stephen S. Amon, et. al., the lethal dose for humans, extrapolated from monkey data in [21] and [22], is 0.09-0.15 µg of toxin by iv and im., 0.70-0.90 µg by inhalation and 70 µg orally.

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Figures:

Fig. 1. Schematic structure of botulinum toxin. In the form of single-chain, toxins have relatively little potency as neurotoxins. Neurotoxic activation requires a two-step modification in the tertiary structure of the protein. In the first step, the parent chain is cleaved between amino acids 448 and 449. The result is one light chain (L-chain, amino acids 1-448, approx. 100 kDa) and one heavy chain (H-chain, amino acids 449-1295, approx. 100 kDa) mutually connected by a disulfide bond (-S-S-). The light chain is associated with one atom of zinc (Zn2+). In this form toxin enters the axon terminal. The second activating step, disulfide reduction, occurs only after internalization by the target cell.

*Last Update 8 July 2002