Botulinum Neurotoxin: From Weapon to Cosmetic

Botulinum neurotoxin is the deadliest substance on Earth; a single gram would kill over a million people if inhaled and 8.3 million if injected. So, naturally, humans have developed a specific method to inject it into our faces. Believe it or not, this neurotoxin is the primary component of Botox!

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Meet the Maker: Clostridium botulinum

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Source: wiki.kenyon.edu

C. botulinum actually comprises four diverse groups of microbes, classified together solely due to their ability to produce the botulinum neurotoxin (incidentally, C. baratii and C. butyricum can also produce the toxin) [1]. C. botulinum are rod-shaped, spore-forming, oxygen-hating bacteria. They live in soil and aquatic environments worldwide–probably even in your backyard. They also flourish in damaged or poorly sterilized canned food, but die in temperatures under 12.8°C (55°F) [1,3,9].

Despite carrying the 1 or 2 neurotoxin genes needed to produce botulinum toxin, these microbes are not generally pathogenic. They use the toxin to kill a wide variety of microbial species surrounding them, and then secrete enzymes to break down the proteins and carbohydrates from the corpses in order to utilize their nutrients. These enzymes can even degrade chitin, which is a component of the exoskeletons of arthropods and insects [6].

When under stress, C. botulinum forms spores, a dormant form of the bacteria that enables survival during adverse conditions. These spores are vulnerable to temperatures below 3°C (37°F) and in pHs below 4.6 but can survive standard cooking and food processing procedures and are behind many foodborne cases of botulism (which we will get to shortly)[3]. In fact, the modern technique for canning developed expressly for killing C. botulinum spores [9]! Despite this, conditions which promote the germination of the spores and production of the toxin are rarely found in food or in our digestive tracts. Consequently, the spores often pass through our digestive tracts without ever causing a problem [1,8].

Botulinum Neurotoxin: The Toxin

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The toxin produced by C. botulinum is described as the most poisonous and deadliest substance known to science. It’s earned the status of a Select Agent, which means serious criminal prosecution for anyone violating the strict federal guidelines on obtaining, handing, and documenting it [3]. As previously mentioned, a single gram of the crystalline toxin would kill over a million people if evenly dispersed and inhaled and 8.3 million people if split into injections. For a 70 kg human, the lethal dose of the crystalline toxin is estimated to be 0.09-0.15 µg intravenously, 0.7-0.9 µg through inhalation, and 70 µg orally (1 microgram (µg) = 0.000001 grams (g); in other words, very tiny amounts will kill you). It is tasteless, odorless, and colorless. Luckily, the toxin cannot penetrate human skin and it is easily destroyed; heating it to 85°C (185°F) will detoxify it and it will inactivate 2 days after being aerosolized [1].

Botulinum neurotoxin is a protein that has seven distinct types, designated by the letters A through G. Each requires a specific antitoxin (for example, the antitoxin to A will not treat* the B form), and A, B, E, and (rarely) F cause illness in humans while C and D mostly impact animals [1,7].

The protein itself is “an astonishing modular nanomachine” that combines recognition, trafficking, protein unfolding, translocation, protein refolding, and catalysis in a single molecule [5]. Its toxicity comes from its enzymatic activity as a zinc proteinase; that is, it cuts another protein. The toxin is extremely specific, targeting a protein in the SNARE substrate that is required for neuronal vesicular release of acetylcholine in neuromuscular junctions (bear with me with this one) [1,5].

To translate that into something a little more understandable, the toxin targets the specific location of where your nerves meet your muscles, the synapse of a neuromuscular junction (see the simplified diagram below). When you move a muscle, an action potential travels down your nerve from your brain or spinal cord and triggers the contraction of your muscle. When the action potential reaches the end of the nerve, it causes vesicles (which are little packets containing chemicals) containing acetylcholine to fuse with the cell membrane and release the chemical into the synapse. The trigger for contraction is the binding of the chemical acetylcholine to receptors on the muscle cell. The SNARE complex is a group of proteins required to anchor and fuse the vesicle to the nerve membrane, which is what allows the release of the contents into the synapse. Consequently, the toxin prevents muscle contraction, causing a flaccid paralysis.

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*Unfortunately, the effect of this toxin is irreversible, which contributes to its notoriety as the deadliest substance. The antitoxin will prevent cleaving of SNARE in other synapses, but the damage is permanent until the body grows new axon twigs to reactivate these synapses (see the section on Botulism).

Botulism: The Intoxication

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Source: Wikimedia

Botulism is the condition that occurs when a human becomes poisoned with botulinum neurotoxin. The name is derived from botulus, which means sausage, thanks to a breakout of paralyzing illness that was associated with sausage consumption in the late 18th century [6]. In 1897 it was recognized as being caused by a microorganism, which was consequently named C. botulinum. The poisoning occurs in many animals besides humans: insects living in the soil may pick up the spores and are then eaten by birds, fish ingest the toxin from their watery habitats, and farm animals have been known to die of botulism as well. The numbers of fish and birds impacted by a toxic outbreak can number hundreds of thousands [8]. In humans, the numbers are far tamer. Fewer than 200 cases are reported each year in the USA, while the more unfortunate Europeans end up with over 2500 cases a year [1,6].

There are three main forms of botulism: foodborne, wound, and intestinal (sometimes infant botulism is also included). Intestinal botulism is caused by spores of C. botulinum germinating and releasing toxin in the colon, while foodborne and wound botulism result from ingesting the toxin in foods or through injuries. There is also, unfortunately, a weaponized form of botulism caused by inhaling toxin. The good news is, there is no person-to-person transmission [1,3,6,7].

People suffering from botulism become paralyzed due to the action of the toxin. In all forms, the toxin enters the blood and is carried to neuromuscular junctions, where the toxin binds to the nerves and triggers endocytosis (the cell engulfs the toxin, a bit like Pac Man). Once inside the nerves, botulinum neurotoxin cleaves the SNARE complex as previously described, preventing the nerve from causing muscle contractions. The extent and pace of the paralysis varies between people, taking as little as two hours or as long as eight days to show and ranging from a mild paralysis to an apparent comatose state requiring feeding tubes and ventilation. The initial paralysis symptoms present as a difficulty seeing, speaking, swallowing, and general weakness. It can progress to a loss of head control, hypotonia, limb paralysis, a loss of gag reflex, and death from airway obstruction and an inadequate volume of air in the lungs [1,7].

The classic triad of symptoms is summarized as a symmetric, descending flaccid paralysis along with a lack of fever and a clear sensorium; the toxin cannot enter the brain, so there are no sensory interpretation or cognition effects. The mind is perfectly clear, although the body may appear completely paralyzed [1,3].

As far as recovery and therapy goes, there is no medical cure for botulism. Recovery occurs by the body producing new motor axon twigs that grow from the poisoned nerve to synapse onto the paralyzed muscle and provide a new source of acetylcholine. This can take anywhere from weeks to years, depending on the severity of the case. Supportive care is vital, which includes feeding tubes, ventilation, and the treatment of secondary infections. Antitoxins are used to minimize further nerve damage by the spread of the toxin. Thanks to the supportive care available along with the low dose ingested in most cases of botulism, the fatality rate is only 5-10% [1,6].

Toxin: Biological Warfare

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Naturally, humans have sought to exploit the world’s deadliest substance for morally dubious uses. If there’s one thing humans excel at, it’s finding creative ways to kill each other. Botulinum neurotoxin poses a major biological weapon threat due to its potency, lethality, ease of production, and ease of transport.

Countries (and terrorists) began to develop and use botulinum neurotoxin as a biological weapon about 60-70 years ago. The Japanese biological warfare group, Unit 731, fed cultures of C. botulinum to prisoners during their occupation of Manchuria to study its effects (although, many of the experiments conducted by this unit appeared to be less for scientific advancement and more out of an inexplicably grotesque perversion). In the Second World War, concerns that Germany had begun to develop and concentrate the toxin led the US biological weapons program to produce the toxin and send over a million doses of antitoxin with their troops for the D-Day invasion. It was also developed by the Soviets shortly after, along with attempts to introduce the gene to create the toxin into other species of bacteria [1].

More recently, the Japanese cult Aum Shinrikyō used aerosols filled with the botulinum neurotoxin in three attacks between 1990 and 1995. Luckily, their technique in using aerosol dispersal was flawed and all three attacks failed. Meanwhile, Iran, North Korea, Iraq, and Syria have all developed or are in the process of developing botulinum toxin as a weapon. After the 1991 Persian Gulf War, Iraq admitted to having produced 19,000 liters of concentrated botulinum toxin. This amount is approximately three times the amount needed to kill the entire current human population, and a good portion of it is still unaccounted for [1].

Accordingly, the CDC has a specific surveillance system for human botulism to rapidly detect and react to any outbreak that could possibly be an attack.

Therapeutics: Botox and Medical Uses

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Adapted from: facialsurgeon.tumblr.com & thebirthinglady.com

While part of the world’s population got giddy over utilizing the deadly toxin in weapons, another portion of people promptly set about figuring out how to utilize it for human benefit. Surprisingly, this has given rise to the deadliest substance also being one of the safest human therapeutics, if used correctly by experts. Millions of doses are put to therapeutic purposes each year. The wonderful thing about nature’s toxins is that they are often extraordinarily precise. Botulinum neurotoxin is incredibly specific for peripheral cholinergic nerve terminals, and if certain precautions and doses are used, it does not spread significantly from the site of injection. It also has a high specificity for diseased hyperactive nerve terminals: in diseases that cause excessive muscle contraction, the nerves have an increased rate of vesicular recycling, which increases the uptake of the toxin into the nerve [8].

Botulinum toxin was the first biological toxin to be licensed to treat diseases associated with glandular, smooth, and skeletal muscle overactivity. It is used to help treat spasticity from cerebral palsy, focal spasticity, cervical torticolliis, strabismus, blepharospasm, and other movement disorders. It is also used to reduce pain and to help treat migraines, chronic back pain, strokes, and traumatic brain injury [1,3,4,8].

The key to using the toxin medically is to use an obscenely tiny amount in order to, you know, not kill the patients. The therapeutic commercial product contains 0.3% of the lethal dose. Considering how tiny the lethal dose is (0.09-0.15 µg of the crystalline toxin intravenously, in case you forgot), this concentration is nearly impossible to conceptualize (approximately 0.00027 µg for a 70kg person). Along with the fact that the body develops new synapses to get around the toxin, any therapeutic use tends to require repeated injections of teeny, tiny amounts [3].

Research is also being conducted into using components of botulinum toxin for neuronal repair. Type C botulinum toxin contains three exotoxins, one of which modifies the Rho proteins that are involved in neuronal growth cones that aid neurite and axon growth. One of the Rho proteins, RhoA, stops growth of neurons and induces the collapse of the growth cone. While this occurs naturally in undifferentiated neurons during development and in your adult hippocampus, it is also a result of neuronal injury. Consequently, a fragment of the C type of botulinum toxin is being studied to potentially prevent the collapse of growth cones in injuries, helping with recovery and repair of lesions. As the toxin only affects differentiated neurons (adult neurons with specific functions), it doesn’t harm the natural growth cone collapse in developing and undifferentiated neurons [2]!

Finally, this toxin is also the main component of Botox. Yes, humans go to unbelievable lengths for the sake of vanity. Personally I think I will steer clear of injecting the deadliest substance into my face, whatever the concentration, but if you would like to paralyze wrinkles away, this is the way to do it. Special localization techniques and injections in the hands of experts minimize the spread of the toxin and fix people’s faces and provide a temporary, youthful effect. In a small number of cases, the toxin spreads and causes muscle weakness and botulism. In other cases, people take Botox injections overboard and, well, I’m sure a google image search or a glance through Hollywood’s registry will provide plenty of examples of what can go wrong [4].

References

[1] Arnon, S. et al. “Botulinum toxin as a biological weapon.” JAMA. 2001. 285(8):1059-1071.

[2] Just, I. et al. “Therapeutic effects of Clostridium botulinum C3 exoenzyme.” Naunyn-Schmied Arch Pharmacol. 2011. 838:247-252.

[3] Katona, Peter. “Botulinum toxin: therapeutic agent to cosmetic enhancement to lethal biothreat.” Anaerobe. 2012. 18:240-243.

[4] Lim, E. et al. “Accurate targeting of botulinum toxin injections: how to and why.” Parkinsonism and Related Disorders. 2011. 17:S34-S39

[5] Montal, Mauricio. “Botulinum neurotoxin: a marvel of protein design.” Annual Review of Biochemistry. 2010. 79:591-617.

[6] Peck, M. et al. “Clostridium botulinum in the post-genomic era.” Food Microbiology. 2011. 28:183-191.

[7] Rodloff, A. and Kruger, M. “Chronic Clostridium botulinum infections in farmers.” Anaerobe. 2012. 18:226-228.

[8] Rossetto, O. et al. “Botulinum neurotoxins.” Toxicon. 2013. Pre-print.

[9] Sobel, Jeremy. “Botulism.” Food Safety. 2005. 41:1167-1173.

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3 thoughts on “Botulinum Neurotoxin: From Weapon to Cosmetic

  1. Thank you for this article on Botulism. For further research you can refer to my blog at http://botulismrecovery.wordpress.com
    I am recovering from Iatrogenic Botulism, which is Botulism caused by Botox injections. This drug can be extremely dangerous. Last year there were over 3,000 complaints to the FDA about side effects of Botox. Learn the risks. I wish I had. I am now in a wheelchair and hoping to prevent others from sufffering.

  2. Pingback: Tetanospasmin and Tetanus: The Second Deadliest Toxin | Biogeekery

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