Saturday May 31, 2008 - Vol. VII Issue 4[Download PDF for Printing]
Biotoxins: Part 3
On June 12, 2002,
President George W. Bush signed into law the Public Health and Safety Act of
2002 (PL 107-188) which requires that the Department of Health and Human
Services maintain a list of biological agents and toxins which pose a severe
treat to public safety. The list of
biotoxins, as it appears in the August 23, 2002 Federal Resister, (see also 42
CFR Part 72, Appendix A) is as follows:
perfringens epsilon toxin
The U.S. Center for Disease Control (CDC) lumps several of
these toxins into a broad classification called “Selected Low Molecular Weight
(LMW) Toxins”. These include (1)
conotoxins, (2) Saxitoxin, (3) Tetrodotoxin, (4) the T-2 toxin, and (5)
Diacetoxyscirpenol, as opposed to the other listings which are “Protein
Toxins,” which have a high molecular weight.
Generally speaking, the low molecular weight toxins are not destroyed by
cooking and might be potentially used by a terrorist to contaminate surfaces
and food as they are more stable in the environment, and can be more
potentially inhaled as an aerosol. Four
of the LMW toxins are discussed in this Newsletter.
Some other LMW Toxins recognized by CDC but not appearing on
the Department of Health and Human short list are brevetoxins, palytoxin, and
Conotoxins are neurotoxins derived from marine cone snails
of the genus Conus
that occur in the Indian-Pacific Oceans especially
off the coast of Australia. Cone snails
do not occur naturally off the coast of the United States (Hawaii an exception)
or Europe. The conotoxins are in the
toxin sacs of these predatory snails.
The snails use their venom to immobilize and kill fish, shellfish, and
marine worms. Conotoxins are a complex
group of chemicals made up of typically 12 to 40 amino acid residues forming
compact peptide molecules of which over 2000 different variant combinations are
known. There are probably over 50,000
different conotoxins in existence from perhaps 500 different species of cone
snails. Any cone snail species can
inject a mix of many different conotoxins.
of the most deadly cone snails, venomous tube visible, snail can grow up to 10
cm in length; a typical attack takes place in milliseconds with a 70% fatality
rate to humans. Photo from National
Geographic website, Kerry Matz photographer.
of Conus geographus, from www.Biopix.com
(J.C. Schon photo)
of Conus catus
of Conus textile
of C. catus
and C. textile
shells and other cone shells at
Human deaths have occurred naturally when divers and
fishermen have accidentally stepped on a cone snail, or in the process of
harvesting the snails. The shells are
very attractive, and some shells are worth a small fortune to collectors. About 30 deaths have been documented and
studied. There are probably a lot more
deaths that have not been studied or reported.
Deaths occur by injection of the venom if the snails are handled or
stepped upon, but the venom is also toxic by ingestion of the mollusk.
Conotoxins are classified into six different broad
classifications based on their biological activity (Table 1).
Table 1 Biological Activity of Conotoxins
Inhibits nicotinic acetylcholine receptors at nerves and
muscles. The result is paralysis.
Inhibits voltage-graded sodium channels in muscles. The mechanism is similar to that of
saxitoxin produced from red tide algae and discussed in an earlier PEAC
Inhibits the inactivation of voltage dependent sodium
channels (“delta” slows the inactivation of the sodium channel, “mu” inhibits
the sodium channel.)
Affects the calcium channels associated with nerve impulse
transmission at the neuromuscular junction.
Calcium channels are related to sensitivity to pain.
Inhibits voltage-graded potassium channels, resulting in
Blocks nerve impulses that use glutamic acid rather than
acetylcholine as the neurotransmitter.
The extreme toxicity results from several different classes
of conotoxins acting synergistically by different mechanisms. Some of the toxins by themselves are not
lethal but produce tremors or deaden pain.
Some alpha-conotoxins by themselves are lethal by injection at 0.025
mg/kg or even 0.01 mg/kg of body weight, from mouse injection tests. No information is available in the public
domain on toxicity by inhalation [from http://www.cbwinfo.com/Biological/Toxins/Conotox.html
On a molecular scale, conotoxins differ from other biotoxins
in that they are relatively small, compact peptides made up of 12 to 40 amino
acids held tightly together by disulfide bonds. The disulfide bonding network as well as the order of the
specific amino acids and how they are configured determine the specifically of
Clinical symptoms (based on interviews by H. Flecher in 1935
of people “stung” by Cone snails and published in the Medical Journal of
and later interviews) include
Non Fatal Case (full recovery)
arm and pain
numbness spreading rapidly to involve the entire body, with some cardiac
and respiratory distress
weakness, loss of coordination, drooping eyelids, shallow breathing
nausea, stomach cramps, shortness of breath
without pain (some species produce severe pain and spreading numbness)
symptoms occur almost immediately upon injection
occurs as the result of respiratory and/or cardiovascular collapse.
Supportive care includes artificial respiration
There are severe logistics for a potential terrorist to grow
and harvest cone snails for their toxins.
Our search using the Internet failed to uncover any use of Conotoxins as
a terrorist weapon. There is an
interview report on Soviet research using smallpox virus to produce toxic small
peptide chains similar to “conotoxins” [see http://www.homelandsecurity.org/newjournal/Interviews/displayInterview2.asp?interview=3
The potential threat of terrorist use is there because
Conotoxins are being studied as a source of potential drugs for treating
neurological diseases. In addition, the
amino acid sequence forming the peptide chain of several conotoxins have been
determined, and synthetic combinations of specific conotoxins have been
artificially produced. Patents for
producing selected conotoxins or using them for drugs are published in the open
literature. The introduction of genes
into bacteria, which can be grown to produce the toxins is feasible. The possibility of laboratory theft or
someone with the necessary technology and equipment to manufacture the toxins
As an example of medical use, clinical trials are underway
in Australia using a conotoxin Vc1.1 (drug called ACV1) derived from Conus
to treat neuropathic pain in the treatment of sciatica, shingles,
and diabetic neuropathy. The ACV1 also
appears to accelerate the recovery of injured nerves and tissues [see B.G.
Livett et al, “Therapeutic applications of conotoxins that target the neuronal
nicotinic acetylcholine receptor” Toxicon
2006. pp 810-829, abstract
available on Internet]. Additional
examples on the use of LMW Toxins for development of drugs to treat diseases
and neurological conditions is at the website, http://www.bentham.org/cpps/contabs/cpps6-3.htm
. A synthetic version derived from
omega-conotoxin M VII A has found an application in the analgesic drug
The CDC has issued guidelines on the safe handling on
biotoxins including Conotoxins, which can be viewed at http://www.cdc.gov/OD/OHS/biosfty/bmbl5/sections/SectionVIIIG-ToxinAgents.pdf
Additional Reading on Conotoxins: BioScience
, Vol. 47, No. 3 (Mar., 1997), pp. 131-134
Tetrodotoxin is another of what the U.S. Center for Disease
Control (CDC) classifies as a “Selected Low Molecular Weight (LMW) toxin. Poisoning usually occurs as the result of
eating certain marine fish, in particular organ parts where the toxin is
concentrated. Cooking does not destroy
Test animals injected (1 to 10 micrograms per kg of body
weight) with the toxin develop a rapid onset of excitability, muscle spasm, and
respiratory distress. Death may occur
within 10 to 15 minutes from respiratory paralysis. Humans ingesting seafood containing tetrodotoxin show similar
signs of toxicity, typically preceded by numbness of lips, the face, and
extremities. Other symptoms include
sweating, weakness, tremor, incoordination, cyanosis, hypotension, nausea,
vomiting, diarrhea, and abdominal pain.
Cardiac arrhythmias may proceed complete respiratory failure and
cardiovascular collapse. The person
although paralyzed may be conscious until just before death. Death usually occurs within 4 to 6 hours
after ingestion with a range of 20 minutes to 6 hours. The toxin works by inhibiting the sodium
channel at the nerves and muscles.
[information from CDC website and Wikipedia].
representation of Tetrodotoxin molecule, from http://www.chm.bris.ac.uk/motm/ttx/ttx.htm.
CAS Number: 4368-28-9
Lethal dose (from mouse injection test) is 8 micrograms
per kilogram of body weight. Lethal
oral dose (mouse) is 334 micrograms per kilogram of body weight.
Note that tetrodotoxin is a non-protein.
Tetrodotoxin poisoning is usually associated from eating
pufferfish. The toxin does not come
from the fish itself but is produced by certain bacteria, notably Pseudoalteromonas
, and other bacterial species (e.g. Vibrio alginolyticus
). Pufferfish grown in a laboratory free from
the bacteria do not produce tetrodotoxin unless they are fed food containing
the bacteria. The highest concentration
of tetrodotoxin in pufferfish is in the ovaries, liver, intestines, and skin;
these body parts must be removed before the fish is prepared for eating. The muscular flesh of the pufferfish is
considered free of the toxin.
Nevertheless, in Japan where pufferfish [Fugu, as it is called in Japan,
which is also the genus name for several species of pufferfish] is considered a
delicacy, from 1974 through 1983 there were 646 reported cases of pufferfish
poisoning with 179 fatalities. Sushi
chefs who wish to prepare pufferfish [Fugu] must be licensed by the Japanese
government. A technical article on
tetrodotoxin paralytic poisoning is available (Ahasen et al, Singapore Med
Journal) 45(2) (2004)) at http://www.sma.org.sg/smj/4502/4502a2.pdf
. Photos of different pufferfish species are
available at http://saltaquarium.about.com/od/porcupinepufferphotos/Porcupine_Pufferfish_Photos.htm
Tetrodotoxin is also produced by the bacteria inhabiting
other marine and some terrestrial animals.
The list of animals include the blue-ringed octopus, triggerfish, goby,
anglefish, parrot fish, ocean sunfish, porcupine fish, seastars, starfish,
certain species of crabs, flatworms, sea squirts, several marine snails, ribbon
worms, arrow worms, some poisonous frogs, and some salamanders. The blue-ringed octopus uses tetrodotoxin as
venom for injecting its prey (the venom contains both the bacteria and
toxin). With all the different kinds of
bacteria inhabiting different hosts, one would expect different kinds of
tetrodotoxin. There are different
biotoxins produced by different bacteria, but the name “tetrodotoxin” is
reserved for just one molecule. Other
toxins have been given different names, such as anhydrotetrodotoxin, palytoxin,
manitoxin, etc. Two of them (palytoxin
and maitotoxin) have potencies 100 times that of tetrodotoxin. Palytoxin has been isolated from small
marine organisms of the genus Palythoa.
Maitotoxin has been found in certain fishes associated with ciguatera
representation of Maitotoxin (from J. Chandrasekar, Resonance, May
1996, pages 68-70, Me is an abbreviation for CH3 )
Molecular Formula: C164H256O68S2Na2
CAS Number: 59392-53-9
Molecular Weight: 3422
This molecule holds the record of being the largest
natural and most lethal non-protein, non-peptide product made in nature yet
The lethal dose is (from mouse injection tests) is 50
nanograms per kilogram of body weight.
The toxin is produced by the red tide algae Gambierdiscus
toxicus, but human poisoning is associated with eating tropical reef fish
which are contaminated with the red tide algae. The condition is called “Ciguatera Fish Poisoning”.
Paralysis and death may occur upon ingestion. Recovery time among survivors may take
weeks, months, or even years.
Tetrodotoxin has been blamed for “zombie” poisons in Haiti
[see W.H. Anderson, “Tetrodotoxin and the zombie phenomenon”, Journal of
vol 23 (1) pages 121-126 (1988)].
Tetrodotoxin can be synthesized. The papers are in the open literature. [e.g. Kishi, Y. et. Al., Journal
of American Chemical Society,
vol 94, 1972]. For a general survey of methods of tetrodotoxin synthesis (2004)
Tetrodotoxin in many respects is similar to Saxitoxin, which
is discussed in the December 2007 PEAC Newsletter (see http://www.aristatek.com/Newsletter/0712December/TechSpeak.aspx0
). The toxicity is about the same. Both are sodium ion channel blockers. The difference is saxitoxin poisoning occurs
through eating shellfish and tetrodoxin poisoning usually occurs through eating
fin-type fish, in particular pufferfish.
Cooking does not destroy the toxins.
Both poisons can be artificially synthesized. Both have the same potential to be mass produced and used as a
terrorist weapon, to be disseminated as an aerosol or in food.
Toxin is one of several trichothecene myotoxins which occur naturally in moldy
grains (grains infected with Fusarium
mold). The CDC also classifies it as a “Selected Low Molecular Weight
(LMW) Toxin”. The CDC also implements
T-2 toxin as a potential biological warfare agent [based on a report,
Wannemacher R, Wiener SL. Trichothecene mycotoxins. In: Sidell FR, Takafuji,
ET, Franz DR, editors. Medical aspects of chemical and biological warfare.
Vol.6. Textbook of military medicine, part 1: warfare, weaponry, and the
DC: Office of the Surgeon General at TMM Publications, Borden
Institute, Walter Reed Army Medical Center; 1997. p.
representation of T-2 Toxin, from http://www.cbwinfo.com/Biological/Toxins/T2.html
Synonyms: T 2
mycotoxin; Fusariotoxin T 2; Insariotoxin; Mycotoxin T-2; T-2
HT-2 Toxin, C22H32O8, CAS Number
64943-87-2, a metabolite of T-2 Toxin
T-2 Toxin is a powerful natural blister agent which works
by inhibiting protein synthesis.
About 50 nanograms of T-2 Toxin on skin produces the same blistering
effect as 20 micrograms (=20,000 nanograms) of sulfur mustard.
manner in which T-2 Toxin inhibits protein synthesis has been studied by many
researchers (see summary paper on Fusarium
toxins published by the
European Commission, in 2001; Fusarium
is the name of the mold that
produces the toxin), paper at http://ec.europa.eu/food/fs/sc/scf/out88_en.pdf
. Specifically, T-2 toxin attacks a critical
site on the ribosomal RNA. Ribosomes are
the structures within the cell where proteins are made.
Toxicity Data for T-2 Toxin
A detailed summary on toxicity of T-2 Toxin and other
trichothecene myotoxins can be found by visiting the website, [website citation
from Textbook of Military Medicine
T-2 Toxin is toxic by inhalation, skin absorption,
injection, and ingestion. The chemical
is not as toxic by injection or ingestion compared with tetrodotoxin (mouse
lethal dose, injection, LD50
= 1.6 to 3.8 mg/kilogram of body
weight, a mouse weighs about 20 grams).
By inhalation, LD50
(mouse) = 0.24 to 0.94 mg/kg.
Symptoms of Exposure
Symptoms for skin injury are similar to mustard gas but
appear at about a 400 times lower dose.
These symptoms include blistering of the skin and irritation of the eyes
and throat. The dose required to produce
blistering and eye damage is still well below the lethal dose. Inhalation toxicity is comparable to that of
other blistering agents (Lewisite, Mustard).
Symptoms of inhalation exposure include nasal discharge, throat pain,
cough, shortness of breath, and chest pain; the victim spits blood as a result
of pulmonary and bronchial hemorrhage.
Severe poisoning results in prostration, weakness, jerky movement,
shock, collapse, and death. Onset of
symptoms occurs between seconds up to about 20 minutes of exposure. Treatment includes decontamination with soap
If ingested as in contaminated grain products, symptoms
appear between 8 and 12 hours. These
include vomiting and internal hemorrhages in the alimentary track. The intestines, bone marrow, lymph nodes,
spleen, and thymus are particularly affected.
Severe poisoning results in prostration, weakness, jerky movement,
shock, collapse, and death. Treatment
includes supportive care including removal of ingested toxin with adsorbents
such as superactivated charcoal. The
term “alimentary toxic aleuka”, or ATA, is used to describe the poisoning.
Alimentary toxic aleuka occurred in the USSR during 1941-47
and again in 1952, 1953, and 1955 killing thousands of people; the ATA was
traced to the people eating over-wintered wheat. Symptoms included vomiting, abdominal pain, diarrhea followed by
leucopenia, bleeding from the nose and throat, depletion of the bone marrow,
and fever. Extractions of the suspected
wheat showed toxic dermal effects when applied to the skin of test animals. The ATA poisoning was not conclusively linked
to T-2 Toxin, but the presence of Fusarium
fungus species was
established in the over-wintered wheat, and T-2 toxin and HT-2 toxin was found
in later fungal cultures.
Other outbreaks of ATA occurred in China and India. In one Chinese location, 165 subjects became
ill after consuming rice infected with two species of Fusarium
. An ELISA assay of the suspected rice for T-2
Toxin showed a level of 180 to 420 micrograms per kilogram of rice (see
European Commission paper, cited earlier).
Potential for Terrorist Use
T-2 Toxin and other trichothecene myotoxins are relatively
easy to manufacture. The Fusarium
molds can be grown in large fermentation vessels using grains, barley, rice,
maise, or corn as food. Fusarium molds
are found in the soils in which the grain crops are grown, or the grain could
be inoculated with a particular mold such as Fusarium sporotrichioides
. The yield of T-2 toxin may be several grams
per kilogram of grain material. The T-2
Toxin could be harvested and spread as an aerosol. The target might be people or agriculture (livestock, food
The trichothecene myotoxins including T-2 Toxin are in
general stable compounds which are not destroyed during processing or cooking
of food, and they do not degrade at high temperatures (from Eriksen, G.S.,
1998, cited in European Commission paper).
Diacetoxyscirpenol and other Trichothecene Myotoxins
There is a fairly long list of toxic chemicals produced from
molds, which can affect grain products or the air quality in buildings. One of them, diacetoxyscirpenol, is on the
Department of Health and Human Services list of biological agents and toxins,
which pose a severe treat to public safety.
Two other trichothecene myotoxins are also discussed below.
representation of Saratoxin H, from
Produced by mold Stachybotrys chartarum; the mold
may contaminate water-damaged homes and other buildings (wallboard,
fiberglass, cellulose products, etc.).
LD50 (mouse, injection) = 1.0 to 1.4 mg/kg.
If mold spores, fungal fragments, or toxin is inhaled, it
can cause nosebleeds, chest pain, and pulmonary hemorrhage. Contact with skin may cause rash. May be fatal if ingested or inhaled in
large quantity. A cause of infant
deaths (pulmonary hemorrhage) by chronic exposure to mold spores in
contaminated houses. Other symptoms
headache, fatigue, elevated body temperature.
Additional reading on inhaling fungal fragments inside
1 Bush R.K., Portnoy J.M., Saxton A., Terr
A.I., Wood R.A.: The medical effects of mold exposure.
J Allergy Clin Immunol
2 Brasel T.L., Douglas D.R., Wilson S.C.,
Straus D.C.: Detection of airborne Stachybotrys chartarum
macrocyclic trichothecene mycotoxins on
particulates smaller than conidia. Appl Environ Microbiol
3 Cho S.-H., Seo S.-C., Schmechel D.,
Grinshpun S.A., Reponen T.: Aerodynamic characteristics
and respiratory deposition of fungal
fragments. Atmos Environ
4 Murphy W.K., Burgess M.A., Valdivesio
M., Livingston R.B., Bodey G.P., Freireich E.J.: Phase I
clinical evaluation of anguidine. Cancer
5 Scheel C.M., Rosing W.C., Farone A.L.:
Possible sources of sick building syndrome in a Tennessee
middle school. Arch Environ Health
6 Brasel T.L., Martin J.M., Carriker C.G.,
Wilson S.C., Straus D.C.: Detection of airborne
macrocyclic trichothecene mycotoxins in
the indoor environment. Appl
7 Brasel T.L., Campbell A.W., Demers R.E.,
Fergusen B.S., Fink J., Vojdani A., et al: Detection of
trichothecene mycotoxins in sera from
individuals exposed to Stachybotrys chartarum
representation of Nivalenol., from http://www.biopure.at/biopure-index/datasheets/mdc/NIV.htm.
CAS #: 23282-20-4
Isolated as a white powder, m.p. 222oC
Produced from mold Fasarium nivale, which
contaminates grains. Like T-2 Toxin,
it inhibits protein synthesis via binding to the ribosome but is less toxic.
LC50 (mouse, oral): 39 mg/kg (from European
Commission paper, cited earlier).
Chemist’s representation of diacetoxyscirpenol, from
Sigma-Aldrich website, http://www.sigmaaldrich.com
There is potential for a terrorist to mass produce
diacetoxyscirpenol by fermentation using a starch-rich grain or potatoes as a
Produced from molds of species Fasarium such as
Fasarium sambucinum, F. moniliforme, equiseti, F. graninearum,
etc., which can contaminate grains, potatoes, peas, soybeans, and is toxic if
the food is consumed by people or livestock.
Diacetoxyscirpenol also has been detected in crude building materials.
LD50 (mouse, intravenous injection)= 12
mg/kg. LD50 (rat,
intravenous injection) = 1.3 mg/kg;
LD50 (rat, oral) = 7.3 mg/kg, from http://www.cbwinfo.com/
Primarily a concern with livestock fed moldy food, with
symptoms similar to the T-2 Toxin
The same contaminated grain products may contain
diacetoxyscirpenol, T-2 Toxin, and Nivalenol.