Fungal Toxins

Document Sample
Fungal Toxins Powered By Docstoc
					Mycotoxins
Secondary Metabolites
   Organic compounds which have no direct role in
    major metabolic pathways
   Fungi produce remarkable diversity of these
    secondary metabolites
   May serve to discourage predators or suppress
    competition
   Formation quite specific, often confined to one
    species or just one strain
   Thousands of secondary products from fungi
    have been analyzed and characterized
Secondary Metabolites

   Many have widespread commercial
    importance
   Others have well known health effects
   Included - antibiotics, toxins, alkaloids,
    volatile organic compounds
       Antibiotics toxic to microorganisms
       Fungal toxins harmful to humans or other animals
Fungal Toxins

     Mycotoxins formed by hyphae of common
      molds growing under a variety of
      conditions

     Mushroom toxins formed in the fleshy
      fruiting bodies of higher fungi
Mycotoxins
   Mycotoxins produced by many fungi growing in
    contaminated foods and other substrates.
   Small molecules – low MW
   Generally, the highest levels in substrates with
    high water activity and warm temperatures.
   Can develop in grains or nuts in the field due to
    metabolism of pathogenic or saprobic fungi on
    the living plant
   Forage grass may contain mycotoxins because
    of a pathogenic fungus or a fungal endophyte
Mycotoxins

   More commonly -- mycotoxins develop in storage
    and remain within the food after processing and
    cooking
   Many common indoor environmental contaminants
    are toxigenic - able to produce toxins
   Some studies revealed significant levels of airborne
    mycotoxins in occupational settings, offices, and
    even homes
   Mycotoxins are not volatile so exposure must be in
    airborne spores
Mycotoxins identified in spores

Alternaria alternata        Alternariol
Alternaria alternata        Alternariol monomethylether
Aspergillus fumigatus       Tryptacidin
Aspergillus fumigatus       Fumitremorgen
Aspergillus fumigatus       Verruculogen
Aspergillus fumigatus       Fumiclavine C
Aspergillus flavus          Aflatoxin
Aspergillus parasiticus     Aflatoxin
Fusarium graminearum        Deoxynivalenol, T-2
Fusarium sporotrichioides   Deoxynivalenol, T-2
Stachybotrys chartarum      Satratoxin G, H
Water activity (aw)
   A measure of the moistness of the substrate
   Expressed as a decimal and directly related
    to substrate RH
       If substrate RH = 95%, aw = 0.95
   Water activity of pure water = 1.0
   Fungi can grow under low aw
Low water activities
   Limits to growth 1.0 to 0.55
   Animals function at 0.99
   Many plants wilt at 0.98
   Most bacteria 0.95 and higher (some extreme
    exceptions among halophilic bacteria)
   Fungi down to 0.65
       Xerophilic fungi
       control internal aw by storing glycerol
   Other fungi survive as spores, sclerotia
 Water activities for fungi
aw      Solution            Material        Fungi
1.0     water                            Oomycetes
0.994   blood               meat,veggies    Basidiomycetes
0.98    sea water           wood            and Ascomycetes
0.95    1.5molal NaCl       bread        yeasts (basid)
0.90    2.8molal NaCl       ham          yeasts (asco)
0.85    6.0molal sucro      salami       yeasts, Penicillium
0.75    saturated NaCl      salt fish    Wallemia, etc
0.65    22 molal glycerol                Eurotium(Aspergillus) +
0.55    DNA denatured
Health Effects Of Mycotoxins
   Acute and chronic effects on both humans and
    livestock
   Mycotoxins are believed to be among the most
    potent known carcinogens
   Majority of research focused on health effects
    following ingestion of contaminated food
   Effect range from immediate toxic responses and
    immunosupression to potential long-term
    teratogenic, estrogenic, and carcinogenic effects.
   Possible health effects due to airborne exposure
    (exposure to airborne spores with mycotoxins)
History of Mycotoxins
   Turkey X Disease killed over 100,000 young
    turkeys in 1960 in England
   Affected turkeys stopped eating, became lethargic,
    suffered hemorrhages under the skin, and died
   Autopsies showed livers had undergone extensive
    necrosis, kidneys developed lesions
   Partridges, pheasants, ducklings, and other
    animals also affected
   Only factor in common with all the poisonings was
    Brazilian peanut meal as a feed supplement.
   Toxin isolated from feed associated with fungal
    contaminant Aspergillus flavus
Aflatoxins
   Aflatoxin: A (Aspergillus) - fla (flavus) -
    toxin
   Four toxins soon identified: Aflatoxin B1,
    B2, G1, G2 - blue or green florescence
    under UV-light
   Today known to be 10 aflatoxins
   Aflatoxin B1 most important - highly
    carcinogenic and widespread occurrence
    in foods
Sources of Aflatoxins

   Produced by 3 species of Aspergillus: A.
    flavus, A. parasiticus, A. nomius
   Aspergillus flavus a common saprobe that
    occurs on grains and legumes in storage
   A. parasiticus most toxigenic species
   Aflatoxins not only toxic but also carcinogenic
Aflatoxin Production

    Aflatoxins are produced under certain
     conditions only by some strains
    Nontoxigenic strains of A. flavus used to
     prepare fermented foods in the Orient
Effects of Aflatoxins

    Even when levels not toxic, prolonged exposure
     caused liver cancer in every species of lab
     animal tested
    Believed responsible for high rates of liver cancer
     in population groups in Asia and Africa where
     contaminated food is often consumed
    Toxic effects shown in India in 1974 when
     hundreds were poisoned by eating corn
     containing aflatoxins - 106 people died
    Most important crops - peanuts and corn
Aflatoxin Levels
   Levels permissible in foods subject to legal
    limits in many countries
   Today, foods most frequently contaminated,
    routinely screened before processing or sale
   Permissible limits generally quite low (15-20
    parts per billion)
   Some scientists feel that no detectable levels
    of aflatoxins should be permitted because of
    the carcinogenic effects
Average yearly level of aflatoxin contamination from
corn grown along the coastal plain of Georgia
                                                       ppb
Economic Impact

   Because of enforced limits the presence of
    aflatoxins can have serious economic implications
   In 1980 nearly 66% of random corn samples from
    North Carolina had concentrations exceeding 20
    ppm resulting in a $31 million loss to producers and
    handlers.
   When cows and goats are fed grains contaminated
    with aflatoxins, they produce milk with aflatoxins - As
    a result, limits exist for livestock feed
Aflatoxin B1 is Mutagenic

   Hepatocellular carcinoma (HCC) is a major cause of
    mortality in certain areas of the world
   About 50% of the HCC cases in parts of the world
    where food is contaminated with AFB1show a
    mutation in codon 249 of p53 tumor suppressor
    gene
   Mutation consists of transversion of G->T in the third
    position of codon resulting in serine instead of
    arginine - lab studies confirm
Claviceps purpurea
Ergot of rye
Ergotism

   Ergot contains a number of toxic alkaloids, if
    harvested with the grain and milled into the
    flower - it can cause a disease called
    ergotism
   During Middle Ages called “Dancing Mania”
    and “St. Anthony’s Fire”
   Ergotism can also occur in grazing animals
    that forage on contaminated grain
   Many different alkaloids --- cause many
    different effects
Alkaloids in Ergot

   Gangrenous ergotism - Some of the alkaloids
    constrict blood vessels and impair circulation
       Feelings of burning in calves or intense cold
       In extreme conditions can result in gangrene
       Limbs may drop off or require amputation
   Convulsive ergotism - Other toxins affect the
    CNS
       Hallucinations and convulsions - loss of mental
        function
       Feelings of burning in calves or intense cold
Alkaloids in ergot

   With modern milling techniques, ergotism rare
       Outbreak in France in 1951 - 4 deaths and 150
        hospitalized
       1977 in Ethiopia on contaminated barley
   Salem Witches – may have been ergotism
   Many alkaloids
       ergotamine and ergometrine – cause vasoconstriction
       Lysergic acid amides – affect CNS
   Alkaloids used in medicine – to treat migraine
    headaches and in childbirth
Ergot alkaloids in other fungi

   Recent studies have identified ergot alkaloids
    in other fungi including Aspergillus fumigatus
       Festuclavine
       Derivatives of festuclavine
           Fumigaclavine A
           Fumigaclavine B
           Fumigaclavine C
   These 4 mycotoxins found in A. fumigatus
    conidia
Other Mycotoxins
   Today over 400 mycotoxins have been
    identified from 150 species of fungi with
    new ones discovered each year
   Species of Aspergillus, Penicillium,
    Fusarium, Alternaria, Cladosporium and
    Stachybotrys form mycotoxins.
    Mycotoxin Production
   Can occur in one strain of a species, other strains of
    the same species not toxigenic
   Fungi from air samples in homes tested for
    mycotoxin production using tissue culture of human
    fibroblast cells
   In multiple isolates of a single species (up to 12)
    some produced mycotoxins, others did not – in my
    lab 1/3 isolates were toxin forming
   Warm temperatures and abundant moisture promote
    mycotoxin production
         Aspergillus



         Penicillium




Common Toxigenic Fungi
        Stachybotrys




           Fusarium
Ochratoxins

   Produced by species of Aspergillus such as A.
    ochraceus
   Most important is Penicillium verrucosum which
    occurs on grains
   Ochratoxin A a nephrotoxin responsible for
    nephropathy in pigs and probably humans
   It is immunosuppressive and also assumed to be
    carcinogenic.
    Patulin
   Produced by a number of species of
    Penicillium, Aspergillus and Byssochlamys.
   Most important producer is Penicillium
    expansum.
   Fungus causes a soft rot of apples; toxin
    found in apple juice
   Patulin first attracted attention as an antibiotic
    in 1943; no current interest in antibiotic
    properties.
Trichothecenes

   Produced by several species of Fusarium
   One of the most toxic is T-2
   Believed T-2 responsible for outbreak of
    Alimentary Toxic Aleukia (ATA) in Siberia
    during and after WWII
   In some areas 10% of the population
    developed the disease and in most cases
    it was fatal
Alimentary Toxic Aleukia

   ATA characterized by nausea, vomiting, hemorrhages
    in many organs, bleeding from nose and throat,
    bloody diarrhea, low leukocyte count, exhaustion of
    bone marrow.
   About a third of deaths due to strangulation from
    internal swelling of throat
   Years later scientists made the connection between
    the disease and consumption of moldy grain
   Symptoms appeared when people ate 2 kg of moldy
    grain, 6 kg was lethal. Similar hemorrhagic syndrome
    in animals called moldy corn toxicosis
    Vomitoxin
   Produces vomiting in pigs at low
    concentrations
   Much less toxic than T-2 but is
    immunosuppressive
   Contaminates corn, barley, and wheat
   Permissible limits are 0.3 ppm for flour and
    0.1 ppm in bread or breakfast cereal
   During recent wet growing season, Ontario
    farmers lost $17 million on a wheat harvest
    contaminated with vomitoxin.
Macrocyclic Trichothecenes

   More toxic than T-2
   Produced by Stachybotrys chartarum (S. atra)
    and also by species of Myrothecium.
   Specific toxins are Satratoxins, Verrucarins, and
    Roridins
   Fungi are cellulose decomposers and found
    growing on hay or straw stored under poor
    conditions.
Macrocyclic Trichothecenes

   Responsible for the deaths of many horses, but
    it can also affect cattle, pigs, sheep, poultry
   Complex of unpleasant symptoms like ATA.
   Stachybotrys found indoors in many locations
    growing on ceiling tiles and wallboard
   Concern about airborne inhalation of spores
Yellow Rain
   During the Viet Nam war, there was concern that the Viet Cong were
    using chemical weapons against the US as well as the population in
    Laos and Cambodia
   Victims were sprayed with a yellow rain
   Symptoms were like ATA (possibly some evidence of trichothecene
    toxins in some of the victims. However, the issue was not clear cut)
   Little evidence Viet Cong using chemical weapons
     On September 13, 1981, then-U.S. Secretary of State Alexander
        Haig accused the Soviet Union of supplying trichothecene
        mycotoxins to the Communist regimes in Vietnam and Laos for
        use in counterinsurgency warfare
   Samples of some yellow rain deposits later shown to be largely
    made up of pollen - “cleansing flight theory” – 2002 yellow rain in
    India
   This issue over yellow rain in Viet Nam has never been resolved
    Zearalenone
   Produced by species of Fusarium found in moldy
    corn
   Chronic exposure is estrogenic
       Female pigs especially sensitive causing vulvovaginitis.
       Swelling of the vulva, enlargement of the mammary glands,
        enlargement of the uterus, and vaginal prolapse.
   In lower levels causes infertility, stillbirths, and small
    litters
Fumonisins

    Produced by Fusarium species on moldy corn
    Implicated in cases of esophageal cancer in
     humans and other cancer in animals
Other fungi producing mycotoxins

   Cladosporium – epicladosporic acid –
    implicated in ATA, maybe?
   Alternaria – tenuazonic acid – detected in
    commercial tomato paste
   Pithomyces – sporidesmin – disease in
    sheep
    Summary of health effects of mycotoxins

   Acute and chronic effects on both humans and
    livestock
   Many are potent carcinogens
   Majority of research focused on health effects
    following consumption of contaminated food
   Effects range from immediate toxic responses and
    immunosupression to potential long-term carcinogenic
    effects
   Possible health effects due to airborne exposure
    (exposure to airborne spores with mycotoxins)
Health effects from airborne exposure

   Some epidemiological studies linked
    inhalation of mycotoxins with human disease
   Lack of adequate data on exposure, intake,
    excretion, metabolism
   Little information available on amounts of
    mycotoxins in air
   Experiments with animals show extreme
    toxicity to alveolar macrophages caused by
    several different mycotoxins
Possible associations between inhalation
of specific mycotoxins and disease
Aspergillus flavus       Lung cancer

Aspergillus flavus       Colon carcinoma

Aspergillus flavus       Lung cancer

Stachybotrys chartarum   Malaise

Aspergillus flavus       Lung cancer

Aspergillus ochraceus    Acute renal failure

Aspergillus fumigatus    Tremorgenic encephalopathy
                         (dementia and tremors)

Stachybotrys chartarum   Infant hemosiderosis
Health effects from airborne exposure?

   Clinical studies not completed yet
   Association of Stachybotrys with health effects in
    contaminated buildings but no experimental studies
    with human exposure
   Animal studies suggest effects of respiratory
    exposure very important
   Possible effects: immune suppression, rash,
    headache, fatigue, sore throat, pulmonary
    hemorrhage (in infants), memory loss???
   We need more research studies and data!
Toxic Black Mold in the Media

   Refers to Stachybotrys chartarum
   Media frenzy started with Cleveland baby deaths
    and the initial CDC report in 1997
       CDC retraction in 2000 is seldom mentioned
   Media frenzy stirred up again following the 1999
    lawsuit by Melinda Ballard in Dripping Springs,
    Texas
   Media frenzy periodically re-emerges
Stachybotrys chartarum (syn S. atra and S. alternans)

   Asexual fungus
   Teleomorph has not been identified – recent
    paper using molecular data places teleomorph in
    ascomycete order Hyocreales (form perithecia)
    – possibly genus Melanopsamma
   About 10 to 12 species in the genus
    Stachybotrys
   Memnoniella similar to Stachybotrys, but with
    spores in chains
       Produces similar toxins
       Molecular data indicates it same genus
       Therefore many consider Memnoniella echinata as S.
        echinata
Natural habitat

   Typically a soil fungus, common on
    decaying plant substrates, decomposing
    cellulose (hay, straw), leaf litter, and seeds
   One recent paper indicating it is a pathogen
    on soybean causing root lesions – an
    forming toxins in plant
Stachybotrys chartarum
Indoor locations

   Commonly found indoors on wet materials containing
    cellulose, such as wallboard, jute, wicker, straw, and
    paper
   Also found on wood and wood paneling and on general
    organic debris
   Wet conditions absolutely required
   Hidden in ceiling, walls, floors with little visible evidence
    within the interior of the room but spores can
    contaminate interior through holes or cracks in the
    building materials aided by negative pressure
   Spores can also be transported by air handling system
Spore production
   Dark grey to black (dark
    green, brown) conidia
    produced by cluster of
    cylindrical phialides
   Conidia ovoid about 10-12 mm
    in length
   Conidia roughened to warty to
    ridged when viewed at 1000x
   Conidia produced singly and
    successively into a slime
    droplet that covers the
    phialides.
   Suggested to be dispersed by
    insects in the natural
    environment
   Eventually slime dries and the
    conidia can become airborne
Stachybotrys conidiophore and conidia
Stachybotrys trichothecene toxins

   Macrocylic trichothecenes
       Roridin E and L-2
       Satratoxins F, G, and H
       Isosatratoxins F, G, and H
       Verrucarins B and J
   Trichoverroid trichothecenes
       Trichoverrols A and B
       Trichoverrins A and B
   Satratoxins are generally produced in greater
    amounts than the other trichothecenes, but all
    compounds are produced in low quantities
       They apparently occur in all parts of the fungus
Macrocyclic trichothecenes

   Highly toxic compounds with a potent ability
    to inhibit protein synthesis
   Numerous studies have demonstrated the
    toxicity of toxins from S. chartarum on
    animals and animal and human cells
   Satratoxin is the most cytotoxic of
    trichothecenes tested on mammalian cells,
    even more toxic than T-2 toxin associated
    with ATA
   LD50 in mice for satratoxins is ~1 mg/kg
Other Stachybotrys toxins – non MTR
   Nine phenylspirodrimanes (spirolactones and
    spirolactams) and cyclosporin, which are potent
    immunosuppressive agents
       It has been suggested that the combination of trichothecenes
        and these immunosuppressive agents may be responsible for
        the observed high toxicity of this fungus
   Atranones A-G
   Dolabellane diterpenes
   Stachylysin, a proteinaceous hemolysin (causes pore
    formation and lyses red blood cells)
       Also a hydroxymate siderophore
       It has been suggested these could be pathogenicity factors
        involved in pulmonary hemorrhage in infants exposed to S.
        chartarum.
Non-trichothecene toxins

   These components have a number of
    immune system properties
       Inhibition of TNFa liberation from human
        macrophages
       Inhibition of the complement system
           One of the most common symptoms in moldy buildings
            is recurrent airways infections
           It’s been suggested that metabolites which inhibit the
            complement system may be involved since the
            complement system is an important part of our defense
            against bacteria
Stachybotryotoxicosis history

   1930s in the Ukraine and other parts of Eastern
    Europe, outbreaks of a new disease in horses and
    other animals
       Irritation of mouth, throat, nose
       Shock
       Dermal necrosis
       Decrease in leukocytes
       Hemorrhage
       Nervous disorders
       Death
   In 1938 Russian scientists determined the disease
    was associated with S. chartarum growing on the
    straw and grain fed to the animals
More history
   Intensive studies showed the toxicity of
    Stachybotrys in animals – horses actually fed
    cultures - often resulting in death
   Russians called disease stachybotrytoxicosis
   Disease later reported in other farm animals (cattle,
    pigs, sheep, poultry) from other parts of the world
    but not from N. Am.
   In the late 1930s, the disease reported in humans
    working of farms in Russia. People affected handled
    infected hay or feed
       Symptoms – rash, dermatitis, pain and inflammation of
        mucous membranes, conjunctivitis, chest tightness, cough,
        bloody rhinitis, burning eyes, throat, nasal passages, etc
       Outbreak in Hungary in 1977 and 1996 report in Germany
Toxins identified

   In 1973, Eppley & Bailey were the first to
    isolate trichothecenes from S. chartarum
   They found the known components,
    trichodermol and roridin E and three novel
    components, named satratoxins H, G and F.
   Structures determined 4-7 years later
   Toxins continue to be isolated from
    Stachybotrys
       Atranones and stachylysins identified in past 5
        years
Indoor history

   Croft et al. 1986. Airborne outbreak of trichothecene
    toxicosis. Atmos Ennvir. 20: 549-552
       Over 5 yr period, family complained of headaches, sore
        throats, recurring colds, flu symptoms, fatigue, dermatitis,
        and general malaise
       Air sampling showed S. chartarum spores
       Growing on moist organic material in cold air duct and on
        wood fiber ceiling material
       Home had chronic moisture problems
       Extracts from duct debris had satratoxin H , verrucarin B,
        verrucarin J, and the trichoverrins - also were toxic to test
        animals
       When mold problems corrected – symptoms gone
   Several other reports following this paper
Cleveland Baby Deaths

   1993-1994 cluster of cases of pulmonary
    hemorrhage (hemosiderosis) in infants
   All the homes of the infants had high levels of total
    fungi and S. chartarum (based on air and surface
    sampling)
   Homes had water damage
   Stachybotrys isolates from homes produced
    trichothecenes
   Similar cases published in the late 1990s
   1997 – CDC report suggested an association
More evidence

   Unrelated case of a 7 year old with various
    symptoms – cough, fatigue, fever, recurrent
    pneumonia
   Stachybotrys identified in fluid washed from
    lungs
   His home damaged from a flood and
    Stachybotrys (and other fungi) growing on
    wallpaper near his bedroom
   Child became symptom free when moved
USA Weekend Cover Stories
Controversy

   Can Stachybotrys chartarum cause pulmonary
    hemorrhage? Can it effect human health in indoor
    environments?
   Most feel there is insufficient evidence to prove a
    link
   In 2000 CDC retracted previous statement and
    issued reports critical of the study done in Cleveland
    and concluded the association was not proven
   2004 IOM of NAS link to toxin-related symptoms not
    proven
A lot we don’t know!

   There may be multiple modes of action for
    Stachybotrys to affect human health
   Toxicosis is clearly important but immune-
    suppressive compounds may also be
    important, especially in infants
   Hemolytic compounds also important. Some
    suggest maybe we should consider this a
    pathogen!
Dorr Dearbon, MD - Cleveland

   Dorr Dearborn was one of the physicians
    involved in the Cleveland baby case and has
    continued studies of Stachybotrys
   "There is a negative health impact of living in
    a mold environment…But the details as to
    what the health effects are and how much
    mold it takes – that is what we don't know."
Summary

   Health effects on indoor exposure to
    Stachybotrys not proven
   Data since 1930s suggests that we should
    not be handling material contaminated with
    Stachybotrys without safety equipment
   Indoor environments contaminated with
    Stachybotrys are not healthy – especially for
    children

				
DOCUMENT INFO