NEUROTOXINS-

THE FOOD ANGLE

BY: PRATHAMESH KUDALKAR

13FET1011

Food Engineering and Technology,

Institute of Chemical Technology, Mumbai

CONTENTS

1. Introduction 1

2. Botulinum Toxin 1 2.1 Clostridium Botulinum 1

2.2 The Toxin 2

2.3 Structure 2

2.4 Mechanism 3

2.5 Exposure and Transmission 4

2.6 Food Sources 4

2.7 Prevention of Contamination 5

2.8 Symptoms 5

2.9 Treatment 6

2.10 Detection Methods 6

3. Tetrodotoxin 6 3.1 Poisoning 7

3.2 Symptoms 7

3.3 Treatment 7

4. Conotoxins 8

5. Toxic Metals 8

6. Glutamate and Nitric Oxide 8

7. Conclusion 8 8. References 9

1. Introduction:

Neurotoxins are the substances that are poisonous or destructive to the nerve cells. Neurotoxins are harmful in

most cases but can also be used constructively. They affect the functioning of developing as well as mature

nervous tissues. Neurotoxins can be of plant, animal, mineral or atmospheric origins. They are mostly exogenic

but the term is also used for classifying endogenous compounds which when present in higher concentrations

may prove neurologically toxic.

Neurotoxins inhibit neuron control over ion concentrations across cell membrane or communication between

neurons across a synapse. Neurotoxin exposure causes damage to the central nervous system having adverse

effects such as muscle paralysis, intellectual disabilities, hyperactivity which may eventually lead to death.

Symptoms may appear immediately after exposure or be delayed. They may include limb weakness or numbness,

loss of memory, vision, and/or intellect, uncontrollable obsessive and/or compulsive behaviors, delusions,

headache, cognitive and behavioral problems and sexual dysfunction. Individuals with certain disorders may be

especially vulnerable to neurotoxins.

Some common neurotoxins are tabulated below:

Botulinum Toxin Lead Chlorotoxin

Tetradotoxin Ethanol Conotoxin

Tetanus Toxin Glutamate Arsenic

Nitric Oxide Tetraethylammonia Mercury

One of the most common source of Neurotoxin exposure is food. Further in this report we are going to analyze

the neurotoxins which have food as their source.

2. Botulinum Toxin

Botulinum toxin (BoNT or BTX) is one of the deadliest and well known Neurotoxin. It is produced by bacterium

Clostridium Botulinum. It is a group of neurotoxins consisting of eight distinct compounds, referred to as BTX-

A,B,C,D,E,F,G,H. It causes a rare disease termed as Botulism. LD50 (Lethal Dose 50) value is 1ng making it the

most potent of all the known poisons. 1g is sufficient to kill approximately 30 billion mice. Nowadays, it is also

commercially produced for medical, cosmetic and research use. It is most feared Biological weapon.

2.1 Clostridium Botulinum

Clostridium botulinum is an anaerobic, Gram-positive, spore-forming rod

that produces a potent neurotoxin. Spores of C. botulinum are widely

distributed in soil, sewage, mud, sediments of marshes, lakes and coastal

waters, plants- fruits and vegetables and intestinal contents of animals and

fishes. Cells are sensitive to low pH (<4.6), lower Aw (0.93), and

moderately high salt (5.5%). The spores are heat-resistant (killed at 1150C)

and can survive in foods that are incorrectly or minimally processed.

Spores do not germinate in the presence of nitrate (250 ppm). Foods most

commonly associated are low acid vegetables (green beans, corn, spinach,

asparagus, pepper and mushrooms) and fruits (figs and peaches) and also

fermented, improperly cooked and smoked fish, meat, poultry and fish

eggs in hermatically sealed containers.

2.2 The Toxins

Seven serotypes (A, B, C, D, E, F and G) of botulism are recognized, based on the antigenic specificity of the

neurotoxin produced by each strain. Specificity of toxins is in cleaving the proteins on presynaptic membrane and

the vesicles present in the Neuromuscular junction. Types A, B, E and F cause human botulism. Types C and D

cause most cases of botulism in animals. As they are highly potent toxins only a very small amount (1ng/kg body

weight) is necessary for severe symptoms and even death. The toxins are heat labile and can be destroyed by high

and uniform heat at 950C for 15 minutes or boiling for 5 minutes.

Characteristics of different types of toxins are as shown below. They are clubbed together in groups on the basis

of their similarity of action in nerve cells. Groups I & Group II are toxic to humans.

Groups I II III IV

Neurotoxin Types A, B, F B, E, F C, D G

Gene Location Chromosome Chromosome Bacteriophage

Activity Proteolytic Non Proteolytic Non Proteolytic Proteolytic

Optimum

Temperature

35-40 18-25 15 ND

Minimum aw 0.94 0.97 ND ND

Minimum pH 4.6 5.0 ND ND

D100 (min) 25 <0.1 0.1-0.9 0.8-1.12

D121 (min) 0.1-0.2 <0.001 ND ND

*The thermal death points given are for the spores that form the particular type of toxin

2.3 Structure

The toxin is in the form of a non-covalently bound complex that contains

several nontoxic proteins that consist of hemegglutinin and

nonhemegglutinin, and weighs 150 kDa. In order for this polypeptide

molecule to become toxic, it is cleaved by a protease at one-third the

distance from the N terminus.The exact enzyme that performs this

function has still yet to be been determined. This action yields two

fragments: a smaller, lighter fragment weighing 50 kDa and a heavier

fragment with a larger weight of 100 kDa. Proteolytic activity is located

at the N-terminal end of the light chain of botulinum toxin

2.4 Mechanism

Normal Functioning:

To thoroughly understand the mechanism of botulinum toxin, it is imperative to first understand the normal

neurotransmitter release function in the body.

The above image shows the enlarged view of Neuromuscular junction. Acetylcholine plays an essential role in the

body. It is responsible for regulating the somatic nervous system, which controls the voluntary movements of the

skeletal muscles, and is the only of its kind. It is carried by synaptic vesicles to the presynaptic membrane, where

it is released by exocytosis. This released acetylcholine binds to Acetylcholine receptors on the muscle cell and

brings about the contraction of muscle. The exocytosis is facilitated by group of proteins termed as SNARE

proteins. SNARE proteins consist of Synaptobrevin which is bound to the vesicles while SNAP-25 and Syntaxin

are bound to the presynaptic membrane. These proteins form a Synaptic Fusion Complex between vesicle and

membrane allowing the release of acetylcholine.

Action of Botulinum Toxin:

Once it enters the body through food or other sources, Botulinum Neurotoxin neurotoxins target the body’s

peripheral nervous system. After entering the body, it is transported by blood stream to the nerve cells. They pass

through the presynaptic membrane of motor nerve terminals and enter into the neuron cell by endocytosis. The

heavier chain forms a channel within the membrane in which the lighter chain can then pass through into the

cytoplasm. The botulinum toxin produces specified cleaving proteases that allows it to successfully attach to the

synaptic vesicles. The carboxy-terminal domain of the heavy chain recognizes a specific binding site, while the

nitrogen-terminus transports the lighter chain into the nerve cytosol. The lighter chain contains metalloproteases

that target SNARE proteins involved in controlling the exocytosis machinery. Neurotoxin types depend upon the

characteristic of lighter chain. SNARE proteins are group of proteins which facilitate the release of acetylcholine

from Synaptic vesicles. SNARE proteins consist of Synaptobrevin which is bound to the vesicles while SNAP-

25 and Syntaxin are bound to the presynaptic membrane. Type B,D,F,G cleaves Synaptobrevin while type A,C,E

cleaves SNAP-25. In addition, type C also cleaves Syntaxin. Thus membranes of vesicles carrying acetylcholine

and presynaptic membranes do not fuse, in turn blocking the exocytosis of neurotransmitter acetylcholine.

If acetylcholine is absent, muscle contraction cannot take place resulting in regional flaccid paralysis of muscle.

It affects the cardiovascular as well as respiratory muscles. Death is caused mainly due to failure of respiration.

2.5 Exposure and transmission

Foodborne botulism

Infant botulism

o It occurs when infants ingest Clostridium botulinum spores, which germinate into bacteria that

colonize in the gut and release toxins.

o In most adults and children older than about six months, this would not happen because natural defense

that develops over time prevent germination and growth of the bacterium.

Wound botulism

o Wound botulism is rare and occurs when the spores get into an open wound and are able to reproduce

in an anaerobic environment.

Other types of intoxication.

o Water borne

o Airborne

2.6 Food Sources

The spores are heat-resistant (killed at 1150C) and can survive in foods that are incorrectly or minimally processed.

Foods most commonly associated are low acid vegetables (green beans, corn, spinach, asparagus, pepper and

mushrooms) and fruits (figs and peaches) and also fermented, improperly cooked and smoked fish, meat, poultry

and fish eggs in hermatically sealed containers.

2.7 Prevention of Contamination.

Clostridium Botulinum is an anaerobic in nature, so its growth is mainly found in canned foods. Only reliable

method to destroy Clostridium Botulinum is by heat sterilization. All the canned food must be properly sterilized

before it is marketed.

2.8 Symptoms

At the initial stage (generally 12h to 36h, but can be 2h), some gastrointestinal symptoms (nausea, vomiting,

diarrhea and constipation) are evident. Neurological symptoms appear in a short time particularly when the

amount of toxin consumed is more, which includes blurred or double vision, difficulty in swallowing, breathing

and speaking, dryness of the mouth, and paralysis of different involuntary muscles that spreads to the lung and

heart.

2.9 Treatment

The person who has been intoxicated by botulinum toxin should be given supportive care for breathing like

Ventilator. Equine antitoxin must be administered within 18 hrs. Even if extremely small dosage is ingested,

antitoxins must be taken immediately or it may lead to paralysis.

2.10 Detection Methods

Mouse bioassay was the earliest method used for detection of Botulinum toxin.

Modern Methods:

ELISA

PCR

HPLC

DNA Microarrays

Real time PCR

3. Tetrodotoxin

Tetrodotoxin, frequently abbreviated as TTX, is a

extremely toxic neurotoxin. It is found mainly in

fishes such as pufferfish, porcupinefish, ocean

sunfish etc. TTX is found in

the liver, gonads, ovaries, intestines, and skin of

these fish. LD50 value for humans is 25mg/kg.

Tetrodotoxin intoxication is found mainly in Japan.

3.1 Poisoning

The toxin can enter the body by ingestion, injection, or inhalation, or through abraded skin. The mechanism of

toxicity is through the blockage of fast voltage-gated sodium channels. These are required for the normal

transmission of signals between the body and brain. As a result, TTX causes paralysis of voluntary muscles

(including the diaphragm and intercostal muscles, stopping breathing), loss of vagal regulation of heart rate

(causing it to increase to around 100bpm), and loss of sensation etc.

3.2 Symptoms

The diagnosis of pufferfish poisoning is based on the observed symptomology and recent dietary history.

Symptoms develop within 30 minutes after ingestion and include paresthesia of the lips and tongue is followed

by hyper-salivation, sweating, headache, weakness, lethargy, incoordination, tremor, paralysis etc. Death usually

occurs in 4-6 hours if proper treatment is not provided.

3.3 Treatment

Therapy is supportive. If ingested, treatment can consist of emptying the stomach, feeding the victim activated

charcoal to bind the toxin, and taking standard life-support measures to keep the victim alive until the effect of

the poison has worn off.

4. Conotoxins

Conotoxins are found mainly in creatures having shells like snail, oyster and sometimes even crabs. It inhibits the

activity of Ca channel. Some Conotoxins were found to have different activity. Cases of Conotoxin intoxication

have not been reported yet or misreported. Nowadays, Conotoxins are permitted by FDA for their use as Pain

Killers. Not much research has been done on Conotoxins.

5. Toxic Metals

Metal contamination in food is easily possible during cultivation, harvesting and processing. Mercury (Dimethyl

mercury) is the most potent toxin ever.Aluminium and Mercury are capable of inducing CNS damage by

migrating into brain by crossing brain, bone barrier. Arsenic is widely found in the soil. It is destructive towards

cytoskeleton. Lead is a potent neurotoxin whose toxicity has been recognized for at least thousands of years. Lead

results in increased absorption of calcium in cell leading to apoptosis (cell death).

6. Glutamate & Nitric Oxide

Glutamate and Nitric Oxide are widely used in food as an additives. Glutamate and Nitric Oxide are part of CNS

and hence termed as endogenous neurotoxins. Nitric oxide (NO) is commonly used by the nervous system in

inter-neuron communication and signaling. Glutamate is its functions as an excitatory neurotransmitter. It is only

when these endogenous compounds become highly concentrated that they lead to dangerous effects. Increased

concentrations lead to DNA damage, swelling and apoptosis. Also, NO is carcinogenic.

7. Conclusion

Most of neurotoxins go unnoticed in routine food tests. The only full proof way to avoid proliferation of

neurotoxins in food is by following Good Manufacturing Practices (GMPs). Utmost precautions must be taken

while choosing raw materials for food production. Use of additives must strictly align with the guidelines given

by the control and monitoring authorities.

8. References

Olney, John W. (2002) "New Insights and New Issues in Developmental Neurotoxicology."

Adams, Michael E., and Baldomero M. Olivera (1994) "Neurotoxins: Overview of an Emerging Research

Technology."

BOTULINUM TOXIN Mechanisms of action Dirk Dressler , Fereshte Adib Saberi, Egberto Reis Barbosa

Botulinum Toxin Author: Divakara Kedlaya, MBBS

Lebeda et al (2008), Onset Dynamics of type A botulinum neurotoxin induced paralysis

Kiernan, Matthew C., Geoffrey K. Isbister, Cindy S.-Y. Lin, David Burke, and Hugh Bostock (2005)

"Acute Tetrodotoxin-induced Neurotoxicity after Ingestion of Puffer Fish."

Arnon et al (2001) "Botulinum Toxin as a Biological Weapon."

Jacob, Reed B., and Owen M. McDougal (2010) "The M-superfamily of Conotoxins: a Review."

James Jay, Food Microbiology

Other Miscellaneous Sources.