A brief introduction to Mycotoxins
Dr.Kedar Karki
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
toxic to microorganisms Fungal toxins harmful to humans or other animals
Antibiotics
Fungal Toxins
Mycotoxins
formed by hyphae of common molds growing under a variety of conditions toxins formed in the fleshy fruiting bodies of higher fungi
Mushroom
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
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 Alternaria alternata Aspergillus fumigatus Aspergillus fumigatus Aspergillus fumigatus Aspergillus fumigatus Aspergillus flavus Aspergillus parasiticus Fusarium graminearum Fusarium sporotrichioides Stachybotrys chartarum
Alternariol Alternariol monomethylether Tryptacidin Fumitremorgen Verruculogen
Fumiclavine C Aflatoxin Aflatoxin Deoxynivalenol, T-2
Deoxynivalenol, T-2 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
Other fungi survive as spores, sclerotia
fungi control internal aw by storing glycerol
Xerophilic
Water activities for fungi
aw
1.0 0.994 0.98 0.95 0.90 0.85 0.75 0.65 0.55 water blood sea water 1.5molal NaCl 2.8molal NaCl 6.0molal sucro saturated NaCl 22 molal glycerol DNA denatured
Solution
Material
Oomycetes meat,veggies Basidiomycetes wood and Ascomycetes bread yeasts (basid) ham yeasts (asco) salami yeasts, Penicillium salt fish Wallemia, etc
Fungi
Eurotium(Aspergillus) +
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
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
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
Produced by 3 species of Aspergillus: A.
Aflatoxin Production
are produced under certain conditions only by some strains Nontoxigenic strains of A. flavus used to prepare fermented foods in the Orient
Aflatoxins
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 (1520 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
Years 1977 1978 1980 1981 1983 1984 1990 1994 Concentration of Aflatoxin (ng/g) ppb 622 57 204 37 128 37 217 6
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
A Fumigaclavine B Fumigaclavine C
Fumigaclavine
These 4 mycotoxins found in A. fumigatus conidia
Other Mycotoxins
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.
Today
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
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
by Fusarium species on moldy corn Implicated in cases of esophageal cancer in humans and other cancer in animals
Produced
Other fungi producing mycotoxins
implicated in ATA, maybe? Alternaria – tenuazonic acid – detected in commercial tomato paste Pithomyces – sporidesmin – disease in sheep
Cladosporium – epicladosporic acid –
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
Aspergillus flavus
Possible associations between inhalation of specific mycotoxins and disease
Lung cancer
Aspergillus flavus Aspergillus flavus Stachybotrys chartarum Aspergillus flavus
Aspergillus ochraceus
Colon carcinoma Lung cancer Malaise Lung cancer
Acute renal failure
Aspergillus fumigatus
Stachybotrys chartarum
Tremorgenic encephalopathy
(dementia and tremors)
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
Stachybotrys Memnoniella similar to Stachybotrys, but with
spores in chains
About 10 to 12 species in the genus
Produces similar toxins Molecular data indicates it same genus Therefore many consider Memnoniella echinata as S. echinata
Natural habitat
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
Typically
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
Stachybotrys conidiophore and
conidia
Stachybotrys trichothecene toxins
Macrocylic trichothecenes
Trichoverroid trichothecenes
Roridin E and L-2 Satratoxins F, G, and H Isosatratoxins F, G, and H Verrucarins B and J
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
Atranones A-G Dolabellane diterpenes Stachylysin, a proteinaceous hemolysin (causes pore formation and lyses red blood cells)
It has been suggested that the combination of trichothecenes and these immunosuppressive agents may be responsible for the observed high toxicity of this fungus
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
of TNFa liberation from human macrophages Inhibition of the complement system
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
One
Inhibition
Stachybotryotoxicosis history
1930s in the Ukraine and other parts of Eastern Europe, outbreaks of a new disease in horses and other animals
In 1938 Russian scientists determined the disease was associated with S. chartarum growing on the straw and grain fed to the animals
Irritation of mouth, throat, nose Shock Dermal necrosis Decrease in leukocytes Hemorrhage Nervous disorders Death
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
years
and stachylysins identified in past 5
Indoor history
Croft et al. 1986. Airborne outbreak of trichothecene toxicosis. Atmos Ennvir. 20: 549552
Several other reports following this paper
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
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 immunesuppressive 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