Amatoxins

Justin Hessinger

Tess Demeter

What is a toxin?A toxin is a poisonous substance that is

produced by one organism that has negative effects on another organism

They include small molecules, peptides, or proteins

Why did we choose amatoxins?

No, but really, we felt that amatoxins (alpha-amitin) were interesting because they are the cause of over 100 human deaths annually worldwide. The organisms that are affected by these seemingly harmless shrooms suffer mild symptoms hours after ingestion that completely disappear only to kill their unexpecting victims days later.

Cause they’re freakin’ cool!!!

What are amatoxins?

Class of toxins produced by certain types of Amanita species of mushrooms

Bicyclic octapeptide derivativeszymogenic until they reach the host

There are at least 10 known variants of amanitin toxin

Amatoxins account for 95% of fatal mushroom poisoning

Where does amanitin come from?

Alpha-Amanitin is produced by some of the mushrooms from the genus Amanita.

The most common/deadly species:

Death cap mushroom(Amanita phalloides)

Destroying Angel(Amanita virosa)

Where does amanitin come from?

There are around 600 known species, some of which are actually edible

They are distributed throughout the world and found in regions such as;MexicoCentral AfricaEurope CanadaSouth AmericaUnited States

Mechanism: Inhibition of

RNAPII Alpha-amanitin causes inhibition of RNA

Polymerase II by:strong hydrogen bonding to the bridge

helix on RNA Polymerase referred to as pore 1.

penetrates at pore1 and regulates translocation and stops elongation at or before translocation occurs.

it does not affect NTP loading, rather it prevents the bridge helix from flexing thus preventing DNA translocation.

Mechanism: Inhibition of RNAPII

Mechanism: Induced

Apoptosis Inhibition of RNA Polymerase II induces stress

activated kinases.

Phosphorylation of p53 protein fraction on serine residues

This causes suppression of p53 degredation

The accumulated p53 are translocated to mitochondria and this increases permeability of the outer membrane which causes the release of cytochrome C to the cytosol.

This causes programmed cell death.

Mechanism: Reactive Oxygen SpeciesAmanitin toxicity is also associated

with an increase in the production of reactive oxygen species through lipid peroxidation

Increases hepatotoxicity

Although many studies note this reaction during amanitin toxicity, they are unclear in stating how amanintin itself induces this response

Types of AmatoxinsThese are all very similar in structure,

often only differing by 1 R-group.

• α-amanitin• β-amanitin • γ-amanitin • ε-amanitin• Amanullin

• Amanullinic Acid • Amaninamide • Amanin•Proamanullin

Intraspecific Variability Toxicity depends on many factors:

Amount of toxin ingested Age; multi-organ failure between 6-8

days ranges10-20% adults22-50% children

Immune Function Exposure Pathway; majority of exposure

is through oral administration, but there are cases of intravenous injection.

Interspecific VariabilityAlthough amatoxins are

extremely toxic to humans and difficult to treat, there are species that are immune to these toxins. The mechanisms of their immunity are highly misunderstood

Caenorhabditis elegans

Drosophila melanogaster

Theoretical Mechanisms of

ImmunityStructural mutations of RNAPII Alpha-amanitin is unable to bind in the

proper locationMost plausible

Metabolic degradationThere are 3 possible modes of

degredation although the most plausible is by cytochrome P450’s

Behavioral Avoidance

Host Immunity

Why are the mushrooms that develop these amatoxins not affected by them?

Protein precursers are usually used when the protein is potentially harmful to the host, but needs to be available on short notice and in large quantities.

The active form of the toxin is not present in the host organisms.

When entering a target species activation occurs by cleavage of the 8 terminal amino acids present on the toxin

Enzymatic reaction catalyzed by prolyloligopeptidase

Exposure pathways

The most common pathway is through oral ingestion

The toxin may be isolated and injected peritoneally or intravenously, although this method is really only reserved for lab research and due to ethicalities, humans are not generally the test subjects

Signs and Symptoms

For humans:6-12 hours: incubation (no observable

adverse effects)Starts with gastrointestinal symptoms

Vomiting, diarrhea, abdominal pain, hypoglycemia, and dehydration

48-72 hours: evident hepatocellular damage Coagulopathy and acute liver failure

6-8 days: acute liver and kidney failure Haemorrhaging, encephalopathy, coma,

and death

Survival ratesAbout 10% of individuals exposed to

amanitin in will die in the U.S.

World wide, about 50% of those exposed to aminitin toxin will die due to inadequate and/or less accessible medical care

Of those that survive, severe liver and kidney damage is extremely common and some patients may die due to complications pertaining to organ failure

Potential TreatmentsSilybin

Comes from the Milk Thistle (Silybum marianum)

Competes with amanitin for transport systems in the plasma membranes in cells

Causes a decrease in penetration of amanitin into hepatocytes

Thioctic AcidPowerful antioxidant Inhibits free radical formation through lipid

peroxidationPrevents further oxidative tissue damage

Potential TreatmentsBenzylpenicillin

Inhibits trasporting polypeptide 1B3 in the plasma membrane which impedes amanitin penetration of hepatocytes

N-acetylcysteineUnclear mechanismMay inhibit hepatic uptake of amanitinMay act as strong antioxidant

CimetidineListed as a treatment, but there was no

known mechanism found

Potential Treatments To date it seems that the most effective treatment

involves pumping the stomach right after eating the toxin. Because this treatment is extremely time dependant and

symptoms may take hours to show up, it’s not largely reliable.

Although there are other proposed treatments, the ones that we have included are the most common across notable sources.

The only treatment other than emptying the stomach content, that has shown any potential benefits through decrease in hepatic necrosis is Silybin.These results, as noted in various studies, can be limited

or nullified by the experimental conditions.There are other studies that have resulted positively with

different toxins, but themselves list a variety of limitations and problems with their research

How do you know?Before eating a random mushroom,

there are a few things you can do:

Meixner Test: tests for presence of amanitin in the mushroomLimitations: cannot differentiate between

toxic and hallucinogenic species because psilocin gives a false positive

Mezler’s Reagent: tests for spores of amyloid, non-amyloid, and dextranoidUsed to identify different types of fungi

Other uses??There is potential for therapeutic use

against pancreatic carcinomaThrough conjugation of Amanitin to Anti-

epithelial cell adhesion molecules (EpCAM), antitumor properties have been shown

EpCAM’s are over expressed in many cancers.

The conjugated molecules are able to bind these markers and initiate apoptosis

ConclusionsAlthough amatoxins are widely

distributed and extremely toxic, they are still very misunderstood.

The toxic effects suffered from exposure to amatoxins are difficult to treat and the mechanisms behind their action is still unclear. Despite these difficulties, continued research and understanding is very important because they may be the key to future treatments for other illnesses and survivorship.

So remember kids,

be careful when picking your shrooms!!!

As we understand that peer-reviewing is a time-consuming process and you may be tired, here is some legal fun you can have with some awesome shrooms. ENJOY!!!http://onlinesupermario.com/mario-world.php

Literature CitedBrueckner, F. and Cramer, P. 2008. Structural basis of transcription inhibition by α-amanitin and implications for

RNA polymerase II translocation. Nature Structural & Molecular Biology. Vol. 15, No. 8: 811-818

Madalan, J., Piotrowska, A., Gomulkiewicz, A., Sozanski, T., Podhorska-Okolow, M., Szelag, A., and Dziegiel, P. 2011. Benzylpenicyllin and acetylcysteine protection from α-amanitin-induced apoptosis in human hepatocyte cultures. Experimental and Toxicologic Pathology. Vol 63: 311-315.

Moldenhauer, G., Salnikiv, A., Luttgau, S., & Harr, I. (2012). Therapeutic potential of amanitin-conjugated anti-epithelial cell adhesion molecule monoclonal antibody against pancreatic carcinoma. JNCI, 1-13.

Nikolova, G., Karamalakova, Y., Hadjibojeva, P., Georgiev, T., Tolekova, A., Gadjeva, V., and Zheleva, A. 2010. Severe mushroom toxin alpha amanitin causes generation of reactive oxygen species in liver tissues of mice – A comparative study by two different instrumental methods. Trakia Journal of Sciences. Vol. 8, No. 2: 149-154.

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mushroom poisoning syndromes. (2012). Retrieved from http://www.namyco.org/toxicology/poison_syndromes.html