Botulinum Toxin

Overview


Botulinum toxin is among the most deadly naturally occuring Neurotoxins, produced by the naturally occurring Clostridium botulinum and causing the fatal disease Botulism. Minute doses of the toxin can be fatal and according to Arnon et al, "a single gram of crystalline toxin, evenly dispersed and inhaled, would kill more than 1 million people" (#Arnon et al, 2001).  The Center for Disease Control classify botulinum as one of the six highest-risk threat agents of bioterrorism. Despite its impressive toxicity, the botulinum toxin, marketed under the trade name "Botox", has a variety of cosmetic and medical uses including (#Schiavo et al, 2000, #Dickerson and Janda, 2006, #Dressler and Benecke, 2007). The most popular use of botox is to remove face wrinkles through paralysis of facial muscles (#Kuczynski, 2004).

Chemical Description


Botulinum toxin is a protein consisting of seven related A-B toxins. Each botulinum toxin molecule is comprised of a heavy chain and of a light chain, connected by a disulfide bond.

Mechanism


Botulinum toxin is a "blocking agent" preventing the release of certain neurotransmitters, specifically Acetylcholine, from the endings of the motor nerves  (#Kennedy, 2002 and (#Kent, 1998).

Botulinum toxin has a light chain and a heavy chain, each of which contributes to the toxicity. The heavy chain allows the protein to bind to and enter a neuron. After the heavy chain allows entry, the light chain acts like a protease and cleaves proteins that would normally allow neurotransmitters to leave the cell. This is essentially a disruption of exocytosis or the release of neurotransmitters (#Dong et al. 2007). The blocked neurotransmitter, Acetylcholine, normally transmits a nerve impulse to a muscle, signaling the muscle to contract. By blocking this neurotransmitter, botulinum toxin causes its characteristic flaccid paralysis (#Madigan and Martinko, 2006).

Uses


Medical Uses
Main Article: Botox
The botulinum toxin has beneficial medical uses including various cosmetic uses, treatment of cerebral palsy, urinary incontinence, and even for smiles that are deemed "too gummy" (#Scholtes et al, 2008, #Franco, 2007, and #Polo, 2008).


From #Polo, 2008.

The flaccid muscular paralysis that can be fatal in botulism is used for an advantage in medical treatments as the toxins are injected into the muscles, at different sites on the body often the face , which leads to temporary paralysis (effects lasting from 3 to 9 months) of the wrinkle-causing muscles (#Alex Kuczynski, 2004).

Deaths have rarely been associated with the therapeutic uses of botulinum toxin. Fatality could arise when when the toxin spreads beyond the injection site and leads to respiratory palysis from Botulism (#Dressler and Benecke, 2007).

BioWeapon
Security analysts rank the use of botulinum as a high threat just after the use of anthrax on the list of organisms that could be used as a bioweapon (#Kennedy, 2002). The development of a vaccine for botulinum, considered the single most poisonous substance known, are underway (#NIGMS, 2007).

Adverse Health Effects


Main Article: Botulism
Botulism
The botulinum toxin, produced by the bacterium Clostridium botulinum, causes the illness Botulism most commonly from consuming food contaminated by the bacteria. Improperly preserved foods accounted for many cases of Botulism, but more recently it has been associated with freshly prepared foods that were not properly refrigerated. Individuals suffering from a mild case of botulism may experience muscular weakness, cramps, vomiting, and diarrhea, while more severe cases lead to peripheral muscular weakness and respiratory paralysis. The type of paralysis caused is a flaccid paralysis, without muscular stiffening or contracting (#Schiavo et al, 2000). The botulinum toxin's most significant adverse health effect is its prevention of neurotransmission, causing paralysis. When death occurs from Botulism, it is generally caused by the paralysis of the respiratory muscles, leading to respiratory failure (#Kent, 1998 and #Madigan and Martinko, 2006).

Routes of Exposure


The botulinum toxin most frequently enters the body through ingestion of foods contaminated by Clostridium botulinum. Infant botulism occurs when an infant swallows Clostridium botulinum spores, which grow inside the infants GI tract. Infant Botulism is commonly associated with eating contaminated honey. Clostridium botulinum can also enter the body through infected wounds. Wound Botulism is associated with intravenous drug use. While botulinum toxin has not been used as a bioweapon, there is a potential that such a weapon could be developed, which would provide another route of exposure (#Dickenson and Janda, 2006, #Madigan and Martinko, 2006, and #Morrison et al, 2006).

Recent Research in Toxicity of Botulinum Toxin


Continued research in the toxicity of the botulinum toxin is essential because of its extreme toxicity, medical uses, and potential threat as a bioweapon. The 2006 article from ACS Chemical Biology, "The Use of Small Molecules to Investigate Molecular Mechanisms and Therapeutic Targets for Treatment of Botulinum Neurotoxin A Intoxication" recognizes these concerns, and argues that a detailed understanding of the molecular mechanism of botulinum toxin is important and needs to be kept up-to-date, and evaluates strategies for combating its toxicity. Intoxication is a multi-stage process that #Dickenson and Janda, 2006 outline as follows:

Binding to the Target Cell and Internalization: Botulinum toxin binds to nerve terminals by their heavy chain domain, and are internalized by receptor mediated endocytosis. Recent research suggests that the receptor used is the synaptic vesicle protein SV2. An addition component of cellular recognition is low-affinity interactions between the toxin and the gangliosides. These two components form the basis for the double-receptor model, where the botulinum toxin must bind to both of these receptors before it is internalized by the cell.

Receptor-Mediated Endocytosis: There are two plausible processes that may explain botulinum toxin receptor-mediated endocytosis: that is is basically identical to how most ligands are internalized to cells, or the process is a retrieval phase of the vesicle recycling mechanism. Most researchers subscribe to the first explanation, but further research is required to be definitive. Dickerson and Janda describe the latest experimental data: "Labeled derivatives of synaptotagmin antibodies have been used to monitor membrane retrieval and reformation of intraneuronal vesicles", which support the second theory. Again, neither explanation is definitive.

Translocation: The currently proposed mechanism involves a pH-dependent structural rearrangement of the toxin that takes place inside an acidic compartment within the cell, which allows the toxin to enter the cytosol. This implies that the substrates for botulinum toxin are in the cytosol, and that the light change protease must leave the endosome. Current research suggests that as pH decreases (as acidity increases) endosomal domains are exposed, and that these domains allow penetration of the lipid bilayer, allowing translocation of the active region to the cytosol. The exact mechanism of membrane penetration is unknown. It has recently been suggested that the heavy chain of the toxin acts as a chaperone protein and a channel.

Inhibition of Neurotransmitter Release: Botulinum toxin acts to prevent exocytosis, specifically the release of Acetylcholine at the neuromuscular junction. Specifically, botulinum toxin cleaves SNARE proteins. SNARE proteins are involved with fusing synaptic vesicles to the plasma membrane. Cleaving of SNARE proteins by botulinum toxin therefore inhibits the release of acetylcholine at the neuromuscular junction, and leads to inhibition of neurotransmission. Cleaving SNARE proteins creates a nonfunctional SNARE complex: in this nonfunctional complex, Ca2+ influx and fusion is disrupted. This is an important aspect to note, because increasing the Ca 2+ concentration in the synaptic terminal can mitigate the effects of botulinum toxin.

Continued research into treatments of intoxication by botulinum toxin is essential, particularly due to its threat as a bioweapon. The current vaccine requires annual boosters to maintain an adequate antibody titer, and the current antitoxins are both equine based and can caused adverse reactions. More research is recommended in the following avenues of treatment:

  • Potassium Channel Blockers
  • Antagonists of Toxin Binding to Target Cells
  • Antagonists of pH-Dependent Botulinum Toxin Translocation
  • Inhibition of the Botulinum Toxin light chain
  • Other therapies with undefined mechanisms

From #Dickerson and Janda, 2006.

Botulism Case Study


The above image is the leg of a man diagnosed with botulism. In the five days before he was admitted to a hospital, he began suffering from nausea, vomiting, blurry vision, speech difficulties, and general weakness. In the two days before he was admitted, he developed double vision and breathing difficulties. Other symptoms present upon hospitalization included paralysis of the eye motor muscles (ophthalmoplegia), drooping eyelids, pupils slow to react to light, facial weakness, and depressed reflexes. A bioassay confirmed the diagnosis of botulism. He was intubated to improve his respiration and received antitoxin treatment, with gradual improvement.

The man was a "skin popping" abuser of black tar heroin. "Skin popping" is a was of using heroin that is both intravenous and subcutaneous. Skin popping black tar heroin is an increasingly common way to contract wound botulism (#Bhidayasiri et al, 2004). This case study is of particular interest as a new route of exposure: the first reported case due to subcutaneous injection was in New York in 1982. As this type of drug use has increased, so has exposure and incidence of botulism. Three quarters of cases worldwide of wound botulism reported were located in California, a state with high abuse rate of black tar heroin (#Merrison et al, 2002).

Risk Prevention


The most important prevention of exposure to the botulinum toxin is practicing good food preparation and hygiene. The botulinum toxin is destroyed by high temperatures; cooking food thoroughly is recommended. Particularly of risk are improperly canned foods. Food stored in metal cans where bacteria are growing may appear bulgy because of the gas produced from bacterial growth. Cans displaying these bulges should be discarded. Infants should not be offered honey. The risk for wound botulism is reduced by seeking medical attention for infected wounds. Intravenous drug use is a risk factor for wound botulism and should be avoided. If botulinum toxin were to be developed as a bioweapon, vaccination could be the best way to reduce risk (#Dickerson and Janda, 2006 and #WHO 2002).

Treatment


Antitoxin should be administered as soon as the clinical diagnosis has been made. Paralysis of the respiratory system may require mechanical ventilation. Wound botulinum may be treated with antibiotics. While vaccinations exist, it is too late to administer them after exposure (#WHO, 2002).


References



Arnon SS, Schechter R, Inglesby TV, Henderson DA, Bartlett JG, Ascher MS, et al. 2001. Botulinum Toxin as a Biological Weapon. JAMA. 285:1059-1070.


CDC (Center for Disease Control and Prevention). 2005. Public Health Image Library (PHIL). Available: http://phil.cdc.gov/phil/home.asp (Accessed 19 March 2008.


Bhidayasiri R,Choi YM, R Nishimura. 2004. Wound Botulism. Postgrad Med J. 80:240


Dickerson JT, Janda KD. 2006. The Use of Small Molecules to Investigate Molecular Mechanism and Therapeutic Targets for Treatment of Botulinum Neurotoxin A Intoxication. ACS Chem Biol. 1(6):359-359


Dressler D, Benecke R. 2007. Pharmacology of therapeutic botulinum toxin preparations. J Disability & Rehabilitation 29:1761-86


Dong M, Tepp WH, Liu H, Johnson EA, Chapman ER. 2007. Mechanism of botulinum neurotoxin B and G entry into hippocampal neurons. J Cell Biol 179(7):1511-22.


Franco I. 2007. Pediatric overactive bladder syndrome: pathophysiology and management. Paediatr Drugs. 9(6):379-90


Kent C. 1998. Basics of Toxicology. New York, NY:John Wiley & Sons, Inc.


Lacy DB, Tepp W, Cohen AC, DasGupta, BR, Stevens, RC. 1998. Crystal structure of botulinum neurotoxin type A and implications for toxicity. Nat Struct Biol 5:898-902


Magidan M, Martinko J. 2006. Biology of Microorganisms. 11th ed. Pearson Prentice Hall


Merrison AFA, Chidley KE, Dunnett J, Sieradzan KA. 2002. Wound botulism associated with subcutaneous drug use. BMJ. 325:1020-1021


Morrison GA, Lang C, Huda S. 2006. Botulism in a pregnant intravenous drug abuser. Anaesthesia 61:57-60


Polo, M. 2008. Botulinum toxin type A (Botox) for the neuromuscular correction of excessive gingival display on smiling (gummy smile). Am J Orthod Dent facial Orthop 133(2):195-203.


Schiavo G, Matteoli M, Montecucco C. 2000. Neurotoxins Affecting Neuroexocytosis. Physiological Reviews 80:717-766


Scholtes VA, Dallmeijer AJ, Becher JG. 2008. Can We Identity Predictors of Multilevel Botulinum Toxin A Injections in Children With Cerebral Palsy Who Walk With a Flexed Knee Pattern?. J Child Neurol.


WHO (World Health Organization). 2002. Botulism Fact Sheet. Available: http://www.who.int/mediacentre/factsheets/fs270/en/ (Accessed 19 March 2008)


National Institutes of General Medical Sciences (NIGMS). "Botulinum Toxin Study May Lead to Inhaled Vaccine"|http://www.nigms.nih.gov/News/Briefs/brief_simpson.htm]. Last updated May 16, 2007. Accessed 4-23-07.


ALEX KUCZYNSKI, "Is It Botox, or Is It Bogus?". December 5, 2004.


Donald Kennedy. "Beauty and the Beast." Science 295 (5560). March 1, 2002. p. 1601.


Reed Abelson, " F.D.A. Approves Allergan Drug for Fighting Wrinkles". April 16, 2002.