Molds, Part 2 by Harriet Ammann More about mold, mold allergies, and toxigenic types of mold by Harriet M. Ammann, Ph.D.

 
The following article is part two of a two-part series article that was written by Harriet M. Ammann, Ph.D., D.A.B.T. She is a senior toxicologist for Washington State Department of Health, Office of Environmental Health Assessments.


Toxicity
Molds can produce other secondary metabolites such as antibiotics and mycotoxins. Antibiotics are isolated from mold (and some bacterial) cultures and some of their bacteriotoxic or bacteriostatic properties are exploited medicinally to combat infections.
Mycotoxins are also products of secondary metabolism of molds. They are not essential to maintaining the life of the mold cell in a primary way (at least in a friendly world), such as obtaining energy or synthesizing structural components, informational molecules or enzymes. They are products whose function seems to be to give molds a competitive advantage over other mold species and bacteria. Mycotoxins are nearly all cytotoxic, disrupting various cellular structures such as membranes, and interfering with vital cellular processes such as protein, RNA and DNA synthesis. Of course they are also toxic to the cells of higher plants and animals, including humans.
Mycotoxins vary in specificity and potency for their target cells, cell structures or cell processes by species and strain of the mold that produces them. Higher organisms are not specifically targeted by mycotoxins, but seem to be caught in the crossfire of the biochemical warfare among mold species and molds and bacteria vying for the same ecological niche.
Not all molds produce mycotoxins, but numerous species do (including some found indoors in contaminated buildings). Toxigenic molds vary in their mycotoxin production depending on the substrate on which they grow (Jarvis, 1990). The spores, with which the toxins are primarily associated, are cast off in blooms that vary with the mold's diurnal, seasonal and life cycle stage (Burge, 1990; Yang, 1995). The presence of competitive organisms may play a role, as some molds grown in monoculture in the laboratory lose their toxic potency (Jarvis, 1995). Until relatively recently, mold poisons were regarded with concern primarily as contaminants in foods.
More recently concern has arisen over exposure to multiple mycotoxins from a mixture of mold spores growing in wet indoor environments. Health effects from exposures to such mixtures can differ from those related to single mycotoxins in controlled laboratory exposures. Indoor exposures to toxigenic molds resemble field exposures of animals more closely than they do controlled experimental laboratory exposures. Animals in controlled laboratory exposures are healthy, of the same age, raised under optimum conditions, and have only the challenge of known doses of a single toxic agent via a single exposure route. In contrast, animals in field exposures are of mixed ages, and states of health, may be living in less than optimum environmental and nutritional conditions, and are exposed to a mixture of toxic agents by multiple exposure routes. Exposures to individual toxins may be much lower than those required to elicit an adverse reaction in a small controlled exposure group of ten animals per dose group. The effects from exposure may therefore not fit neatly into the description given for any single toxin, or the effects from a particular species, of mold.
Field exposures of animals to molds (in contrast to controlled laboratory exposures) show effects on the immune system as the lowest observed adverse effect. Such immune effects are manifested in animals as increased susceptibility to infectious diseases. It is important to note that almost all mycotoxins have an immunosuppressive effect, although the exact target within the immune system may differ. Many are also cytotoxic, so that they have route of entry effects that may be damaging to the gut, the skin or the lung. Such cytotoxicity may affect the physical defense mechanisms of the respiratory tract, decreasing the ability of the airways to clear particulate contaminants (including bacteria or viruses), or damage alveolar macrophages, thus preventing clearance of contaminants from the deeper lung. The combined result of these activities is to increase the susceptibility of the exposed person to infectious disease, and to reduce his defense against other contaminants. They may also increase susceptibility to cancer (Jakab et al., 1994).
Because indoor samples are usually comprised of a mixture of molds and their spores, it has been suggested that a general test for cytotoxicity be applied to a total indoor sample to assess the potential for hazard as a rough assessment (Gareis, 1995).
The following summary of toxins and their targets is adapted from Smith and Moss (1985), with a few additions from the more recent literature. While this compilation of effects does not describe the effects from multiple exposures, which could include synergistic effects, it does give a better idea of possible results of mycotoxin exposure to multiple molds indoors.

* Vascular system (increased vascular fragility, hemorrhage into body tissues, or from lung, e.g., aflatoxin, satratoxin, roridins)
* Digestive system (diarrhea, vomiting, intestinal hemorrhage, liver effects, i.e., necrosis, fibrosis: aflatoxin; caustic effects on mucous membranes: T-2 toxin; anorexia: vomitoxin.
* Respiratory system: respiratory distress, bleeding from lungs e.g., trichothecenes
* Nervous system, tremors, incoordination, depression, headache, e.g., tremorgens, trichothecenes.
* Cutaneous system : rash, burning sensation sloughing of skin, photosensitization, e.g., trichothecenes * Urinary system, nephrotoxicity, e.g. ochratoxin, citrinin.
* Reproductive system; infertility, changes in reproductive cycles, e.g. T-2 toxin, zearalenone.
* Immune system: changes or suppression: many mycotoxins.
It should be noted that not all mold genera have been tested for toxins, nor have all species within a genus necessarily been tested. Conditions for toxin production varies with cell and diurnal and seasonal cycles and substrate on which the mold grows, and those conditions created for laboratory culture may differ from those the mold encounters in its environment.
Toxicity can arise from exposure to mycotoxins via inhalation of mycotoxin-containing mold spores or through skin contact with the toxigenic molds (Forgacs, 1972; Croft et al., 1986; Kemppainen et al., 1988 -1989). A number of toxigenic molds have been found during indoor air quality investigations in different parts of the world. Among the genera most frequently found in numbers exceeding levels that they reach outdoors are Aspergillus, Penicillium, Stachybotrys, and Cladosporium (Burge, 1986; Smith et al., 1992; Hirsh and Sosman, 1976; Verhoeff et al., 1992; Miller et al., 1988; Gravesen et al., 1999). Penicillium, Aspergillus and Stachybotrys toxicity, especially as it relates to indoor exposures, will be discussed briefly in the paragraphs that follow.
Penicillium
Penicillium species have been shown to be fairly common indoors, even in clean environments, but certainly begin to show up in problem buildings in numbers greater than outdoors (Burge, 1986; Miller et al., 1988; Flannigan and Miller, 1994). Spores have the highest concentrations of mycotoxins, although the vegetative portion of the mold, the mycelium, can also contain the poison. Viability of spores is not essential to toxicity, so that the spore as a dead particle can still be a source of toxin.
Important toxins produced by penicillia include nephrotoxic citrinin, produced by P. citrinum, P. expansum and P. viridicatum; nephrotoxic ochratoxin, from P. cyclopium and P. viridicatum, and patulin, cytotoxic and carcinogenic in rats, from P. expansum (Smith and Moss, 1985).
Aspergillus
Aspergillus species are also fairly prevalent in problem buildings. This genus contains several toxigenic species, among which the most important are, A. parasiticus, A. flavus, and A. fumigatus. Aflatoxins produced by the first two species are among the most extensively studied mycotoxins. They are among the most toxic substances known, being acutely toxic to the liver, brain, kidneys and heart, and with chronic exposure, potent carcinogens of the liver. They are also teratogenic (Smith and Moss, 1985; Burge, 1986). Symptoms of acute aflatoxicosis are fever, vomiting, coma and convulsions (Smith and Moss, 1985). A. flavus is found indoors in tropical and subtropical regions, and occasionally in specific environments such as flowerpots. A. fumigatus has been found in many indoor samples. A more common aspergillus species found in wet buildings is A. versicolor, where it has been found growing on wallpaper, wooden floors, fibreboard and other building material. A. versicolor does not produce aflatoxins, but does produce a less potent toxin, sterigmatocystin, an aflatoxin precursor (Gravesen et al., 1994). While symptoms of aflatoxin exposure through ingestion are well described, symptoms of exposure such as might occur in most moderately contaminated buildings are not know, but are undoubtedly less severe due to reduced exposure. However, the potent toxicity of these agents advise that prudent prevention of exposures are warranted when levels of aspergilli indoors exceed outdoor levels by any significant amount. A. fumigatus has been found in many indoor samples. This mold is more often associated with the infectious disease aspergillosis, but this species does produce poisons for which only crude toxicity tests have been done (Betina, 1989). Recent work has found a number of tremorgenic toxins in the conidia of this species (Land et al., 1994). A. ochraceus produces ochratoxins (also produced by some penicillia as mentioned above). Ochratoxins damage the kidney and are carcinogenic (Smith and Moss, 1985).
Stachybotrys chartarum (atra)
Stachybotrys chartarum (atra) has been much discussed in the popular press and has been the subject of a number of building related illness investigations. It is a mold that is not readily measured from air samples because its spores, when wet, are sticky and not easily aerosolized. Because it does not compete well with other molds or bacteria, it is easily overgrown in a sample, especially since it does not grow well on standard media (Jarvis, 1990). Its inability to compete may also result in its being killed off by other organisms in the sample mixture. Thus, even if it is physically captured, it will not be viable and will not be identified in culture, even though it is present in the environment and those who breathe it can have toxic exposures. This organism has a high moisture requirement, so it grows vigorously where moisture has accumulated from roof or wall leaks, or chronically wet areas from plumbing leaks. It is often hidden within the building envelope. When S. chartarum is found in an air sample, it should be searched out in walls or other hidden spaces, where it is likely to be growing in abundance. This mold has a very low nitrogen requirement, and can grow on wet hay and straw, paper, wallpaper, ceiling tiles, carpets, insulation material (especially cellulose-based insulation). It also grows well when wet filter paper is used as a capturing medium.
S. chartarum has a well-known history in Russia and the Ukraine, where it has killed thousands of horses, which seem to be especially susceptible to its toxins. These toxins are macrocyclic trichothecenes. They cause lesions of the skin and gastrointestinal tract, and interfere with blood cell formation. (Sorenson, 1993). Persons handling material heavily contaminated with this mold describe symptoms of cough, rhinitis, burning sensations of the mouth and nasal passages and cutaneous irritation at the point of contact, especially in areas of heavy perspiration, such as the armpits or the scrotum (Andrassy et al., 1979).
One case study of toxicosis associated with macrocyclic trichothecenes produced by S. chartarum in an indoor exposure, has been published (Croft et al., 1986), and has proven seminal in further investigations for toxic effects from molds found indoors. In this exposure of a family in a home with water damage from a leaky roof, complaints included (variably among family members and a maid) headaches, sore throats, hair loss, flu symptoms, diarrhea, fatigue, dermatitis, general malaise, psychological depression. (Croft et al, 1986; Jarvis, 1995).
Johanning, (1996) in an epidemiological and immunological investigation, reports on the health status of office workers after exposure to aerosols containing S. chartarum. Intensity and duration of exposure was related to illness. Statistically significant differences for more exposed groups were increased lower respiratory symptoms, dermatological, eye and constitutional symptoms, chronic fatigue, and allergy history. Duration of employment was associated with upper respiratory, skin and central nervous system disorders. A trend for frequent upper respiratory infections, fungal or yeast infections, and urinary tract infections was also observed. Abnormal findings for components of the immune system were quantified, and it was concluded that higher and longer indoor exposure to S. chartarum results in immune modulation and even slight immune suppression, a finding that supports the observation of more frequent infections.
Three articles describing different aspects of an investigation of acute pulmonary hemorrhage in infants, including death of one infant, have been published recently, as well as a CDC evaluation of the investigation (Monta?a et al., 1997; Etzel et al., 1998; Jarvis et al., 1998; MMWR, 2000; CDC, 1999). The infants in the Cleveland outbreak were reported with pulmonary hemosiderosis, a sign of an uncommon of lung disease that involves pulmonary hemorrhage. Stachybotrys chartarum was shown to have an association with acute pulmonary bleeding. Additional studies are needed to confirm association and establish causality.
Animal experiments in which rats and mice were exposed intranasally and intratracheally to toxic strains of S. chartarum, demonstrated acute pulmonary hemorrhage (Nikkulin et al. 1996). A number of case studies have been more recently published. One involving an infant with pulmonary hemorrhage in Kansas, reported significantly elevated spore counts of Aspergillus/Penicillium in the patient's bedroom and in the attic of the home. Stachybotrys spores were also found in the air of the bedroom, and the source of the spores tested highly toxigenic. (Flappan et al., 1999). In another case study in Houston, Stachybotrys was isolated from bronchopulmonary lavage fluid of a child with pulmonary hemorrhage. (Elidemir et al., 1999), as well as recovered from his water damaged-home. The patient recovered upon removal and stayed well after return to a cleaned home. Another case study reported pulmonary hemorrhage in an infant during induction of general anesthesia. The infant was found to have been exposed to S. chartarum prior to the anesthetic procedure (Tripi et al., 2000). Still another case describes pulmonary hemorrhage in an infant whose home contained toxigenic species of Penicillium and Trichoderma (a mold producing trichothecene poisons similar to the ones produced by S. chartarum) as well as tobacco smoke (Novotny and Dixit, 2000)
Toxicologically, S. chartarum can produce extremely potent trichothecene poisons, as evidenced by one-time lethal doses in mice (LD50) as low as 1.0 to 7.0 mg/kg, depending on the toxin and the exposure route. Depression of immune response, and hemorrhage in target organs are characteristic for animals exposed experimentally and in field exposures (Ueno, 1980; Jakab et al., 1994).
While there are insufficient studies to establish cause and effect relationships between Stachybotrys exposure indoors and illness, including acute pulmonary bleeding in infants, toxic endpoints and potency for this mold are well described. What is less clear, and has been difficult to establish, is whether exposures indoors are of sufficient magnitude to elicit illness resulting from toxic exposure.
Some of these difficulties derive from the nature of the organisms and the toxic products they produce and varying susceptibilities among those exposed. Others relate to problems common to retrospective case control studies. Some of the difficulties in making the connection between mold exposures and illness are discussed below.
Limitations in Sampling Methodology, Toxicology, and Epidemiology of Toxic Mold Exposure. Some of the difficulties and limitations encountered in establishing links between toxic mold contaminated buildings and illness are listed here:

* Few toxicological experiments involving mycotoxins have been performed using inhalation, the most probable route for indoor exposures. Defenses of the respiratory system differ from those for ingestion (the route for most mycotoxin experiments). Experimental evidence suggests the respiratory route to produce more severe responses than the digestive route (Cresia et al., 1987) * Effects from low level or chronic low level exposures, or ingestion exposures to mixtures of mycotoxins, have generally not been studied, and are unknown. Effects from high level, acute sub-acute and sub-chronic ingestion exposures to single mycotoxins have been studied for many of the mycotoxins isolated. Other mycotoxins have only information on cytotoxicity or in vitro effects.
* Effects of multiple exposures to mixtures of mycotoxins in air, plus other toxic air pollutants present in all air breathed indoors, are not known.
* Effects of other biologically active molecules, having allergic or irritant effects, concomitantly acting with mycotoxins, are not known. * Measurement of mold spores and fragments varies, depending on instrumentation and methodology used. Comparison of results from different investigators is rarely, if ever, possible with current state of the art.
* While many mycotoxins can be measured in environmental samples, it is not yet possible to measure mycotoxins in human or animal tissues. For this reason exposure measurements rely on circumstantial evidence such as presence of contamination in the patient's environment, detection of spores in air, combined with symptomology in keeping with known experimental lesions caused by mycotoxins, to establish an association with illness.
* Response of individuals exposed indoors to complex aerosols varies depending on their age, gender, state of health, and genetic make-up, as well as degree of exposure.
* Microbial contamination in buildings can vary greatly, depending on location of growing organisms, and exposure pathways. Presence in a building alone does not constitute exposure.
* Investigations of patients' environments generally occur after patients have become ill, and do not necessarily reflect the exposure conditions that occurred during development of the illness. All cases of inhalation exposure to toxic agents suffer from this deficit. However exposures to chemicals not generated biologically can sometimes be re-created, unlike those with active microbial growth. Indoor environments are dynamic ecosystems that change over time as moisture, temperature, food sources and the presence of other growing microorganisms change. Toxin production particularly changes with age of cultures, stage of sporulation, availability of nutrients, moisture, and the presence of competing organisms. After-the-fact measurements of environmental conditions will always reflect only an estimate of exposure conditions at the time of onset of illness. However, presence of toxigenic organisms, and their toxic products, are indicators of putative exposure, which together with knowledge of lesions and effects produced by toxins found, can establish association.
Conclusions and Recommendations
Prudent public health practice then indicates removal from exposure through clean up or remediation, and public education about the potential for harm. Not all species within these genera are toxigenic, but it is prudent to assume that when these molds are found in excess indoors that they are treated as though they are toxin producing. It is not always cost effective to measure toxicity, so cautious practice regards the potential for toxicity as serious, aside from other health effects associated with excessive exposure to molds and their products. It is unwise to wait to take action until toxicity is determined after laboratory culture, especially since molds that are toxic in their normal environment may lose their toxicity in laboratory monoculture over time (Jarvis, 1995) and therefore may not be identified as toxic. While testing for toxins is useful for establishing etiology of disease, and adds to knowledge about mold toxicity in the indoor environment, prudent public health practice might advise speedy clean-up, or removal of a heavily exposed populations from exposure as a first resort.
Health effects from exposures to molds in indoor environments can result from allergy, infection, mucous membrane and sensory irritation and toxicity alone, or in combination. Mold growth in buildings (in contrast to mold contamination from the outside) always occurs because of unaddressed moisture problems. When excess mold growth occurs, exposure of individuals, and remediation of the moisture problem must be addressed.

Author
Harriet M. Ammann is a senior toxicologist for Washington State Department of Health, Office of Environmental Health Assessments. She provides support to a variety of environmental health programs including ambient and indoor air programs. She has participated in evaluations of schools and public buildings with air quality problems, and has presented on toxic effects from air contaminants, indoors and out, effect on sensitive populations, and other health issues throughout the state. Through her work, she has developed an interest in the toxicology of mold as an indoor air contaminant, and has published and presented on mold toxicity relating to human health.
References for this article are located at: http://www.doh.wa.gov/ehp/oehas/mold.html