Botulinum Toxin: From Poison to Medicinal
by Jiri Patockaa and Miroslav Splinob
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
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  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 . 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.
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 , which produce type
F toxin and C. butyricum , 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 . The toxins affect a broad
range of vertebrate species, but the evolutionary utility of toxin production
to the bacterial host organisms is unclear.
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 . 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
The Structure of Botulinum Toxin and Mechanism of
toxins are synthesized as single-chain polypeptides with a molecular
Wight of approximately 150 kDa . The complete amino acid sequences
for the various serotypes are known  and regions of sequence homology
among the serotypes suggest that all employ similar mechanism of biological
action . 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 . 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  (Fig.
1). In this form, the toxin enters the axon terminal. The second activating
step, disulfide reduction, occurs only after internalization by the
are potent blockers of synaptic transmission in peripheral cholinergic
nervous system synapses, thereby causing paralysis . 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 . The
protease activity of the toxins is confined to their light chains .
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 . A novel combination of theoretical approaches
was exploited to predict which amino acid residues of various botulinum
neurotoxin serotypes participate in forming ion channels . 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 .
Clinical Features of 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 . 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 . 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 . 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
. 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 . Administration of
trivalent or heptavalent botulinum antitoxin is recommended as the treatment
for Botulinum toxin poisoning (in USA, CDC - Center for Disease Control,
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 . As many as 17 countries
were suspected to include or to be developing biological agents in their
offensive weapons programs in 1996  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 . In addition,
before the Aum Shinrikyo used sarin in the 1995 terrorist attack on
the Tokyo subway system, the cult had produced botulinum toxin .
use of botulinum toxin as a possible biological weapon began at least
60 years ago . 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 . 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 .
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 .
Botulinum toxin was one of several agents tested at the Soviet site
Aralsk-7 on Vozrozhdeniye Island in the Aral Sea . 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 . 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 . 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 . 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 . It is noteworthy that
Iraq chose to weaponize more botulinum toxin than any other of its known
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
. In the US, the CDC maintains a well-established surveillance system
for human botulism based on clinician reporting that would promptly
detect such events .
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 . The use of botulinum toxin as
a medicinal agent for correcting cholinergic disorders is very promising
 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 . 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.
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.
- 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.
- . 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 . 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  and , 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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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
*Last Update 8 July 2002