Re: Mycotoxins in Indoor Environments Harriet M. Ammann

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Mycotoxins in Indoor Environments

Harriet M. Ammann1

> 1Senior Toxicologist, Air Quality Program

> Washington State Department of Ecology

>

> http://www.doh.wa.gov/SBOH/Meetings/Meetings_2006/2006-

> 03_08/documents/Tab09c-MycotoxinsIndoors.pdf

>

>

>

> Abstract

> Exposure to mycotoxins produced by toxigenic molds growing in damp

> indoor spaces has been difficult to assess. Monitoring methods

limit

> the characterization of inhalation exposure of any bioaerosol,

> especially that of mycotoxins. Biomarkers promise better ability to

> determine mycotoxin exposures 1.) through direct measures of toxins

> and their products in human tissues, 2.) through immunochemical

> methods, and 3.) measures of effect through novel approaches, e.g.,

> proteomics or genomics. This paper summarizes both the problems

> inherent in measuring exposures and some of the promising methods

> that could help to resolve the current impasse.

> Introduction

> Reports of illness among occupants of damp buildings have become a

> concern, especially with respect to both fungi and bacteria that

> colonize damp spaces. While much illness can be explained as

> allergic sensitization, a number of complaints involving the

immune,

> respiratory and nervous system have implicated mycotoxins.

> Epidemiological studies have been able to relate

building " dampness "

> as the lowest common denominator among studies that differ in

> exposure parameters to asthma and other allergic respiratory

> disease. Such relationships have not been clearly established for

> immune and nervous system effects due to a small number of studies,

> small statistical power of existing studies, and difficulty in

> assessing agents such as mycotoxins.

> Buildings become moist because of leaks in plumbing, roofs, walls,

> windows, or foundations, through flooding, or due to condensation

in

> areas that lack sufficient ventilation. Certain toxigenic fungi,

> such as species of Aspergillus, Penicillium, Stachybotrys,

> Memnoniella, and Chaetomium grow frequently in damp indoor

> environments under conditions in which they can produce mycotoxins

> (1, 2). The various fungi and other microorganisms such as

bacteria,

> yeasts, and protozoa form an ecosystem that changes over time as

> moisture and nutrient levels and activity of the microorganisms

> change. Competition for available ecological niches also can

> increase production of mycotoxins.

> Mycotoxins are well-known agents of disease in animals and humans

> who consume them in foods in sufficient amounts. Inhalation of

> toxins associated with grain dusts and other organic dusts are

known

> to be a problem for workers who have developed pulmonary

> mycotoxicoses (including, but not limited to organic dust toxic

> syndrome or ODTS). Establishing that illnesses seen in workers and

> other occupants of damp indoor environments are caused by

inhalation

> of mycotoxins has been more difficult. This paper examines reasons

> 1

> why this has occurred and explores means of determining better

> estimates of exposure to indoor (mold and) mycotoxins.

> Methods

> Information regarding health effects from exposure to mycotoxins

> produced by toxigenic molds commonly found in damp indoor spaces is

> reviewed. Evidence for considering other exposure agents besides

> spores of toxigenic fungi is examined, and the lack of correlation

> of toxins with disease in epidemiological studies is explored.

> Methods for identification of mycotoxins in environmental media and

> within human tissues are discussed. Biomarkers and broadening the

> concept to markers of toxic effect are considered.

> Discussion

> Symptoms Reported from Occupants of Moldy Buildings

> The most frequent complaints of occupants of moldy buildings are of

> cough, rhinosinusitis and worsening of asthma, and increased

> susceptibility to respiratory and other infections. Such infections

> last longer than " normal, " recur and are often recalcitrant to

> treatment as occurs in immune-compromised patients. Complaints of

> extreme fatigue, headache, memory problems, difficulty in

> concentrating or thinking clearly, numbness and other nervous

system

> effects are also not rare. Complaints overlap toxic, allergenic and

> irritative symptoms, and are difficult to assign to specific agents

> found in buildings (3).

> Molds that grow in damp buildings, and a number of bacteria,

> especially Streptomyces species, have been implicated in studies

> addressing signs of allergy, such as increase in inflammatory

> markers, or increased immunoglobin titers (3). Allergic reaction to

> molds can explain such signs and symptoms in atopic people, while

> those who are not atopic often have illnesses (3, 4), that are

> likely due to other causes such as toxicity. Allergy also does not

> explain increased susceptibility to infection and nervous system

> effects. These are in keeping with mycotoxicoses in herd animals

> consuming moldy feed.

> Problems with Measuring Mycotoxin Exposure Indoors

> Inhalation has generally been considered the route of exposure from

> indoor mold contamination. Because molds such as Aspergillus and

> Penicillium colonize new habitats by casting their spores into the

> air, inhalation of airborne spores has been thought to be the

> primary means of mycotoxin exposure in damp buildings. However,

> mycotoxins are not found only in and on spores (1, 5). They are

also

> found in hyphae and hyphal and spore fragments, which are

> considerably smaller than whole spores or conidia. Mycotoxins are

> secreted into the substrate on and in which molds grow, and are

> found on dust from substrate (1, 5, 6, 7, 8) as well as in small

> airborne particles generated directly by the organisms (6, 10).

> Settled

> 2

> carpet dusts from damp buildings contain mycotoxins (7) as do dusts

> from ventilation systems (8, 9).

> A recent study of aerosolization of fungal propagules using a newly

> designed aerosolization chamber, determined that fragments of molds

> are released from infested ceiling tiles in much greater numbers

> than spores. Depending on fungal species, velocity of air past

> contaminated surfaces, texture of the surface, and amount of

> vibration, concentration of small particles released may be 320

> times greater than spore concentration (6). These particles are of

> considerably smaller size than spores (1.6 & #956; average for

fragments,

> compared to 2-3.5 & #956; for Aspergillus versicolor spores, and 4-7

& #956; for

> Cladosporium cladosporoides spores) (6). Air velocities as low as

> 0.3 meters/second caused spores and small particles to be released

> (11).

> Inhalation of a mixture of spores, fragments, fungal propagules and

> dust represents a much greater potential for exposing occupants to

> mycotoxins than inhalation of spores alone. Smaller particles, with

> their large aggregate surface area, can adsorb more mycotoxins than

> the same mass of larger spores. Small particles are more easily

> inhaled and deposited in airways and the lung, where toxins can

> cause damage locally, or be absorbed into the bloodstream and

> lymphatic circulation and distributed to target organs. Measuring

> toxin concentration of settled or airborne dust could more

> realistically estimate mycotoxin exposure. However, airborne and

> settled dust samples will reflect different spectra of fungi

because

> of settling characteristics and other factors, so that using both

is

> probably necessary (12). Accurate assessment of exposure is

> essential for establishing links with mycotoxicoses.

> Studies that tried to relate symptoms with spore levels from air

> samples have found little correlation with incidence or severity.

> Researchers often conclude that no exposure occurred. Allergic

> symptoms suffered by atopic persons were attributed to mold

> exposure, while similar symptoms suffered by non-atopic individuals

> could not be explained (3), and were dismissed.

> Problems with Determining Mold Exposure

> Problems inherent to characterization of spore concentrations make

> correlation with symptoms of occupants difficult (13). Health

> complaints often predate building investigations, so researchers

> have to attempt to re-construct episodic past exposures. Non-viable

> or viable sampling procedures, usually in only a few locations in a

> building, are performed for short periods of time. Samples are dawn

> by pumps at a few liters per minute for a 5- or 10- minute period.

> Spores captured for non-viable sampling are analyzed via light

> microscopy, and only generic identification can be performed.

> Closely related species such as Aspergillus and Penicillium spores

> cannot be distinguished. Identification to the species level of any

> spore from these genera requires viable sampling. To capture spores

> alive, volumes of air are drawn onto liquid or semi-solid growth 3

> media that contain selective nutrients. Sampling times are limited

> on agar by a need to have spores dilute enough to be separated on

> the surface so that they can be subcultured and identified. In

> either case the process of sampling captures only a few moments in

> the life of airborne spores.

> Spore production is episodic and unpredictable, and periodic taking

> of air samples may miss concentrations of spores that occur with

> blooms, and overcount more buoyant spores that are likely to remain

> airborne longer. Such periodic sampling may also miss occupant

> activities that re-mobilize spores into the air. Whether or not

> spores that are captured are actually enumerated depends on the

> skill of the mycologist/microscopist in identifying visible spores

> in non-viable samples, and on a number of limitations imposed by

> sampling technology, and the biology of the organisms. The choice

of

> growth medium may not support the growth of all species of spores

> captured, since different species have different growth

> requirements. Spores impacting the sides of the sampler may be

> damaged and not grow. Spores may be driven below the surface of the

> growth medium, and may not grow. Molds that can produce mycotoxins

> can inhibit growth of competitors which would not be counted even

> though they may be impacting occupants. Representative sampling of

> what occupants breathe during the day is severely compromised by

> these inherent difficulties.

> Air sampling for spores, regardless of the analytical technique

used

> for counting and identification, underestimates exposure to

> microbes. Since toxins are isolated from captured samples, exposure

> to toxins is also underestimated. Dust sampling for spores gives a

> different spectrum of microbes, and has some usefulness for

> assessing contamination that has occurred over time, but it still

> underestimates genera that are buoyant, and even if all components

> are assayed, will still miss toxic content.

> Extraction of mycotoxins from captured mold spores has been assumed

> to be a way of measuring mycotoxin exposure in most studies.

Usually

> the amount of toxins measured is so small as to be below threshold

> for toxic effect. Because of the problems inherent in capturing

> representative samples of spores from which exposure can be

> determined, and because toxins in smaller particles that are more

> likely carriers of mycotoxins are not characterized, attempts to

> determine toxic cause of human disease indoors have been largely

> unsuccessful.

> Biomarkers of exposure

> The whole problem of exposure assessment of mycotoxins may be more

> usefully addressed through biomarkers. However, since people are

> exposed to certain mycotoxins in food as well through inhalation,

> estimates of exposure indoors must take ingestion exposure into

> account. A number of biomarker techniques have been developed.

> 4

> A few mycotoxins can be detected directly in tissues. Ochratoxin

can

> be measured directly in blood and urine and has been used to

> determine exposure from both ingestion and inhalation. Gliotoxin,

> produced by Aspergillus fumigatus, a mold that is responsible for

> most cases of invasive aspergillosis, is a strong immune

suppressor,

> and is thought to facilitate the invasion of lung tissue by this

> mold. A liquid chromatography-tandem mass spectroscopy (LS-MS-MS)

> assay measured gliotoxin in the lungs and sera of mice infected

> experimentally with A. fumigatus, and in patients with this disease

> (14). Danish researchers have developed a standardized LC-UV-MS

> micro-scale method for screening fungal metabolites and mycotoxins

> from culture extracts, and report that they have been able to

detect

> more than 400 fungal metabolites, including penitrems and macro-

> cyclic trichothecenes (15). However, it is not yet known whether

> these can be detected in human sera or other tissues. Recent work

on

> macro-cyclic trichothecene–producing Stachybotrys chartarum

isolated

> a protein (stachylysin) that could be a marker for exposure, as

well

> as account for an effect (bleeding from respiratory membranes)

(16).

> Aflatoxin adducts, formed when these mycotoxins interact with DNA,

> RNA or protein, can be detected in sera and urine of those exposed

> through a variety of methods. However, DNA-adducts result from DNA-

> repair, and individual variation in ability to repair affected DNA

> may not reflect true exposure or true effect.

> Detection of toxins using immuno-chemical methods has been well-

> addressed for many environmental sampling media, especially for

food

> products and livestock. For instance specific monoclonal antibodies

> against aflatoxins, ochratoxin A, zearalenone, diacetoxyscirpenol

> and T-2 toxin, have been prepared by various researchers through

the

> application of hybridoma technology to mycotoxins (17). Nanogram-

> range concentrations in milk, butter, maize, peanuts, peanut butter

> and porcine kidneys have been detected through the use of enzyme-

> linked or radio-immunoassay or immunoaffinity chromatography.

Assays

> for other toxins, including aflatoxin M1, 3-acetyl deoxynivalenol

> fusarenon X and roridin A have been developed by another laboratory

> (18, 19. 20). Monoclonal antibodies have been developed that are

> capable of detecting fumitremorgin B, produced by Aspergillus

> fumigatus, in rice, buckwheat and corn in the 10 to 60 ng/g range

> (21). Satratoxin G and related satratoxins can be detected in

> environmental samples through an enzyme-linked immunosorbent assay

> in 100 pg/ml concentrations (22). Detection of mycotoxins in an

> exposure medium such as food, or in an air sample can be used to

> estimate nominal exposure and are useful for risk assessment.

> Further development of methods that can measure mycotoxins in human

> sera and tissues would enhance the ability to use such techniques

in

> exposed humans and would eliminate some of the uncertainty from

> extrapolation of exposure from media concentrations, and could be

of

> clinical relevance.

> Monoclonal antibodies against aflatoxin-B1-lysine adducts have been

> used to measure aflatoxin-albumin adducts in human serum collected

> from residents in areas at high risk for liver cancer (23). A

recent

> study of Gambian children

> 5

> discovered that IgA antibodies in saliva were reduced in areas of

> West Africa with high infection-related mortality. Reduction of

> antibodies (and immune defense) may occur through aflatoxin

exposure

> (24).

> Investigators of an outbreak of lung problems in workers repeatedly

> exposed to fungi in a water damaged building (25), used an antibody

> assay for the macrocyclic mycotoxin Roridin A (19, 20). One subject

> among eight developed elevated Immunoglobin G (IgG) antibodies

> against Roridin-hemisuccinate Human Serum Albumin (R-H BSA),

> indicating exposure. However, others thought to have been exposed

> did not develop this immune response (25). The environmental

portion

> of this investigation focused on spores captured in the building,

> and did not consider other airborne sources of mycotoxin exposure.

> Satratoxin-hemisuccinate Bovine Serum Albumin (S-H BSA) has also

> been prepared against the potent toxic macrocyclic trichothecene

> produced by Stachybotrys chartarum (26). Immunoglobin G (IgG)

> antibodies against satratoxin H were measured among patients

exposed

> to a mixture of molds who tested positive for antibodies against

> Stachybotrys chartarum exposure. Both exposure to the mold and

> exposure to the toxin were quantified through serum antibody tests

> (27). A follow-up study examined these patients for damage that

> could account for neuropsychological symptoms and peripheral

> neuropathies such as numbness, tingling and muscle weakness in the

> extremities (28). Patients with documented, measured exposure to

> molds had elevated titers of antibodies (immunoglobulin A,

> immunoglobin A and immunoglobin M, and IgG) to neural-specific

> antigens, such as myelin basic protein, myelin-associated

> glycoprotein, ganglioside GM1, sulfatide, myelin oligodendrocyte

> glycoprotein, alpha-B-crystallin, chondroitin sulfate, tubulin, and

> neurofilament, which are markers of neural damage. They also

> suffered neuropsychological disorders and peripheral neuropathies,

> as determined by tests.

> This series of studies indicates that immunochemical methods may be

> useful in finding the nexus between exposure to mycotoxins and

> related disease. These studies (26, 27, 28) also point in the

> direction of a broader concept of biomarker, which is to include

> markers of effect. The development of techniques such as genomics

> and proteomics has made it possible to develop profiles of gene

> expression that could be related to toxic exposures. A number of

the

> more potent mycotoxins produced by molds commonly found in moisture-

> damaged buildings are potent inhibitors of protein synthesis.

> Measurement of changes in protein profile can indicate exposure to

> specific individual or multiple toxins. Proteomics has been used to

> study the complex nephrotoxicity of OTA. Microarrays were used to

> assess OTA-specific profiles of expression involved in DNA damage

> and apoptosis, response to oxidative stress and inflammatory

> reactions (29).

> Aflatoxins produced most often by Aspergillus flavus and

Aspergillus

> parasiticus, and sterigmatocystin, produced by Aspergillus

> versicolor, are also potent

> 6

> inhibitors of protein synthesis. The trichothecenes are among the

> most potent inhibitors of protein synthesis known. Macrocyclic

> trichothecenes, such as the satratoxins, verrucarins B and J,

> Roridin A produced by Stachybotrys chartarum and Ochratoxin A,

> produced by both Aspergillus and Penicillium species, are all

potent

> inhibitors of protein synthesis (30).

> A number of effects that result from inhibition of protein

synthesis

> can be related to symptoms of occupants of damp buildings (Table

1).

> Key among symptoms that are reported frequently are those that

> affect the immune system (induction of autoimmunity, greater

> susceptibility to infectious disease), neuropsychological deficits,

> headache, nausea, and deficits in memory and concentration. Protein

> synthesis is important for antibody and cytokine production and

> other markers of immunologic effect. Learning and memory depend on

> protein synthesis, and depression of protein synthesis in the brain

> could account for frequent reports of memory and learning deficits

> in mold-exposed patients. The use of proteomics to detect changes

in

> protein patterns as the result of exposure to protein synthesis–

> inhibiting mycotoxins is another tool that could bring more linkage

> between exposure to mycotoxins indoors and effects suffered by

> occupants of moldy buildings.

> Use of measures that assess total toxicity can be used in

> conjunction with mycotoxin assays and environmental contamination

> measures to establish relationships between toxic exposure and

> disease. For instance, total toxicity of captured microbial

material

> (spores, fragments and toxin containing particles) can be

determined

> through toxicity assays such as the methylthiazoltetrazolium (MTT)-

> cleavage test for cellular toxicity. Determination of cytotoxicity

> of the entirety of environmental samples is a first key step in

> determining toxic influence on disease from exposure to damp

> environments (31). Such assays have already been used in clinical

> investigations, although mycotoxin assessment was limited to

> toxicity of captured spores (32).

> Conclusion

> Epidemiological studies, with few exceptions, have been

unsuccessful

> in associating mycotoxin exposure from molds growing indoors. The

> reason for this has been the lack of means to determine toxic

> concentrations in inhaled materials. Attention has focused on

> extracting toxins from spore samples. Yet there is no means of

> measuring actual exposure to spores through the airborne route.

> Inhaled particles of fungal or substrate origin that contain

> mycotoxins are more likely to be agents of exposure, since they

have

> a larger aggregate surface area to which mycotoxins can adsorb, and

> they are more likely to be inhaled and deposited into the lung.

> Such particles, like spores, are likely to have more than toxic

> effects. For instance, they may also be allergenic or irritating to

> mucous membranes. Symptoms of allergy and irritation can complicate

> the presentation of disease

> 7

> since they overlap those caused by toxicity. However, biomarkers

> such as those described in this paper may make toxic exposure

> determination more comprehensive. However, representative

assessment

> of mold species growing in damp indoor environments remains

> tentative, and standardized methods for characterizing air

> concentrations are essential, and underlie determination of toxic

> exposure as well.

> Table 1. Mycotoxins that inhibit protein synthesis and effects.

> MYCOTOXINS AND MECHANISMS

> HEALTH EFFECTS

> Trichothecenes:

> • Inhibit translation of mRNA into protein; highly potent

inhibitors

> of protein synthesis

> • Lipophylic; easily traverse membranes including lung, capillary

> and blood brain barrier

> Inhibition of Protein Synthesis leads to deficits in

> • Learning and memory

> • Autonomic function and other neural function

> • Change Immune response (antibodies, TNFa, macrophage activity,

> etc.) resulting in increase in susceptibility to infectious disease

> and cancer and trigger of autoimmune disorders

> Aflatoxins, sterigmatocystin, Ochratoxin A inhibit protein synthesis

> • At transcription level

> • At translation level

> DNA damage results in

> • Decrease in antibody production, inhibition of immune defenses,

> results increased infectious disease, cancer promotion

> Tremorgens; paralytic mycotoxins

> • Inhibit CNS transmitters, e.g., GABA, Glu, Asp

> Decrease in inhibitory transmitters results in

> • Tremors, hyperexcitability, inco-ordination

> • Paralysis

> Gliotoxin

> • Affects astrocytes

> • Immune modulator

> • Apoptosis inducer: increases

> Decrease in immune defenses and neural changes

> • Facilitates infection by A. fumigatus

> • Affects CNS function

> 8

> caspase-3 activity

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> 11

>