CONSIDERATIONS IN EVALUATING THE EVIDENCE

Most of the information reviewed in this chapter is derived from studies in vitro (that is, studies in an artificial environment, such as a test tube or a culture medium) or animal studies. In vitro studies, as explained below, are not suitable for human risk assessment. Risk can be extrapolated from animal studies to human health effects only if chronic animal exposures have produced sufficient information to establish no-observed-adverse-effect levels (NOAELs) and lowest-observed-adverse-effect levels (LOAELs). Extrapolation of risk exposure from animal experiments must always take into account species differences between animals and humans, sensitivities of vulnerable human populations, and gaps in animal data. Risk assessment requires not only hazard identification but also dose-response evaluation and exposure assessment in humans whose risk is being evaluated. Estimates of exposures of humans to spores, bacteria, microbial fragments, and dust that contains mycotoxins are inherently imprecise and imperfect; biomarkers of exposure to toxins are few, and exposures to single or multiple mycotoxins carried by such agents have not been measured indoors. Thus results of animal studies cannot be used by themselves to draw conclusions about human health effects. However, animal studies are important in identifying hazardous substances, defining their target organs or systems and their routes of exposure, and elucidating their toxicokinetics and toxicodynamics, the mechanisms that account for biologic effects, and the metabolism and excretion of toxic substances. Animal studies are also useful for generating hypotheses that can be tested through studies of human health outcomes in controlled exposures, clinical studies, or epidemiologic investigations, and they are useful for risk assessment that informs regulatory and policy decisions.

BIOAVAILABILITY AND ROUTE OF EXPOSURE

Issues That Affect Bioavailability

Some molds found in damp indoor spaces can produce mycotoxins. Table 4-1 lists a number of mycotoxins and the organisms that produce them. Bacteria can also produce toxins. Although there has not been a large amount of research conducted on the effects of those toxins in the context of their growth in damp buildings, mycotoxins and bacterial toxins have been studied for several decades because of their role in outbreaks of illness associated with the ingestion of moldy food (Etzel, 2002). More recently, concerns that toxins from microorganisms that grow in damp indoor environments may play a role in illnesses reportedly associated with living or working in damp buildings have focused attention on the adverse health effects of inhaling mycotoxins.

TABLE 4-1

Some Mycotoxins and the Microorganisms That Produce Them.

The degree to which a toxin can harm tissues varies with a number of factors, including the chemical nature of the toxin, the route of entry into the body, the amount to which the target organism and organ are exposed, and the susceptibility of the target species (Coulombe, 1993; Eaton and Klaassen, 2001; Filtenborg et al., 1983; Vesper and Vesper, 2002). Interspecies differences in susceptibility can result from differences in absorption, distribution, metabolism, excretion, and the effectiveness of a toxin at its receptor (site of action) (Eaton and Klaassen, 2001; Fink-Gremmels, 1999; Russell, 1996).

Once produced, mycotoxins must be airborne to be inhaled. Mycotoxins are found in and on the spores of molds that produce them, on hyphal fragments, and in dust from substrates on which mold grows and carpet dust (Englehart et al., 2002; Górny et al., 2002; Larsen and Frisvad, 1994; Sorenson, 1993, 1995; Sorenson et al., 1987). They are exuded into the substrate on which a microbial agent is growing, for instance, growth medium in the laboratory and gypsum board, wood, paper, and other building materials in damp or wet buildings (Andersen et al., 2002; Andersson et al., 1997; Buttner et al., 2001; Gravesen and Nielsen, 1999; Nieminen et al., 2002). Mycotoxins have also been isolated from dust sampled in moldy buildings that did not contain mold spores (Englehart et al., 2002; Gravesen et al., 1999; Nielsen et al., 1998). Mycotoxins are found in and on materials that can be aerosolized as particles, so such aerosols can become a source of mycotoxin exposure. But particles are not the only vehicle of exposure to mycotoxins. Mycotoxins are not generally thought to be volatile (Jarvis et al., 1995), but some, such as sesquiterpenes, are semivolatile, and others are at least partially water-soluble and thus able to enter the air in droplet aerosols (Harrach et al., 1982; Peltola et al., 1999, 2002).

The bioavailability of aerosols (including mold spores, contaminated dust, bacteria, and microbial fragments) in the respiratory tract after inhalation depends in part on the size of the particles formed, because their size determines where they are deposited in the respiratory tract and this determines bioavailability. Figure 4-1 shows the relationship between spore diameter and respiratory deposition of a number of mold genera. Figure 4-2 shows the percentage of inhaled spores that are deposited in the respirable (alveolar) area of the lung. The size of mold spores depends on the species that produce them. Spores of genera that use the air pathway for dispersion, including Aspergillus and Penicillium, are in the range of 1–2 µm and are respirable. Some molds (such as Stachybotrys chartarum and Memnoniella echinata) that do not spread their spores through aerosol dispersion are wet and slimy during sporulation; once dry, the spores can be dispersed into air through disturbance of contaminated surfaces and are of inhalable size (5–7 µm) (Sorenson et al., 1987). Such bacteria as Streptomyces californicus isolated from damp indoor spaces are about 1 µm in diameter and can reach the lower airways and alveoli when inhaled (Jussila et al., 2001). Furthermore, Wainman and colleagues (2000) have shown that semivolatile chemicals, such as terpenes and limonene (which can be produced by molds that also produce trichothecene mycotoxins and are also often used indoors as cleaning solvents) react with ozone in indoor air and form particles of a respirable size, 0.2–0.3 µm diameter.

FIGURE 4-1

Spore-deposition coefficients of mold genera in indoor environments. SOURCE: Miller et al., 2001.

FIGURE 4-2

Percentage of inhaled spores that are deposited in respirable (alveolar) area of lung. SOURCE: Miller et al., 2001.

Apart from particle size, determining the bioavailability of mycotoxins found on or in particles is complicated because even toxins from spores that lodge in the nasal mucous membranes can damage cells locally or be absorbed into the systemic circulation (Morgan et al., 1993). Lipid-soluble toxins pass readily through membranes, and the degree of their absorption depends on the blood supply to the tissue (Rozman and Klaassen, 1996).

The bioavailability of mycotoxins and bacterial toxins also depends on residence time and clearance mechanisms. Many mycotoxins affect residence time and clearance by inhibiting phagocytic activity of macrophages or reducing ciliary beat rate (Amitani et al., 1995; Coulombe et al., 1991; Jakab et al., 1994; Sorenson and Simpson, 1986; Sorenson et al., 1986; Wilson et al., 1990). The toxic effect of spores and other particles on alveolar macrophages can impair the ability of these cells to protect against not only mycotoxins but also other bacteria and infectious particles. Slowed ciliary clearance allows longer residence time in the airway and increases the time for absorption of toxins from mold spores, fragments, or dust (Coulombe et al., 1991).

Because the respiratory system is the primary route of entry for gases and particles suspended in air, determination of exposure to air contaminants is complicated because air contains a mixture of substances and the concentration of individual toxicants changes with time and location in the exposure mixture. That is particularly true for toxic compounds originating in microbial contaminants of indoor spaces, because growth and metabolism of microbial organisms introduce additional variables into the exposure paradigm. Difficulties in measuring microorganisms and their products hinder the accurate determination of human exposure to them. Chapter 3 discusses the methods used and the difficulties in measuring such exposures.

TOXIC EFFECTS OF INDOOR MOLDS AND BACTERIA

Exposure to various mold products—including volatile and semivolatile organic compounds and mycotoxins—and components of and substances produced by bacteria that grow in damp environments has been implicated in a variety of biologic and health effects. This section discusses irritation and inflammation of mucous membranes, respiratory effects, immunotoxicity, neurotoxicity, sensory irritation (irritation of nerve endings of the common chemical sense), dermotoxicity, and carcinogenic effects attributed to such exposure.

Mucous Membrane Irritation and Inflammation

Exposure to microorganisms and their products can irritate mucous membranes, such as those of the eyes and respiratory tract, and lead to inflammation via an immune response. Such immune responses are important in normal host defenses, but chronic or excessive release of inflammatory mediators can cause damage to the lung and other adverse effects (Jussila et al., 2003).

Immune responses triggered by exposure to microorganisms and their products include increased production of inflammatory mediators, such as cytokines (for example, tumor-necrosis factor α [TNFα] and interleukin-6 [IL-6]), reactive oxygen species, and, indirectly, nitric oxide (NO) via the induction of nitric oxide synthetase (iNOS) (Hirvonen et al., 1997a,b; Huttunen et al., 2003; Ruotsolainen et al., 1995). Different bacteria evoke different cellular responses. For example, Staphylococcus evokes a response from alveolar macrophages, and Pseudomonas evokes a neutrophil response (Rehm et al., 1980). Mold spores and fragments affect the inflammatory response differently (Hirvonen et al., 1999). A number of in vitro, animal, and human studies that have investigated the irritation and inflammation responses to exposure to microorganisms and molds commonly found in damp indoor spaces are discussed below.

In Vitro Experiments

In vitro experiments use animal or human cell lines or primary cell cultures to explore mechanisms of toxicity for specific target tissues or cells. Although toxic exposure of cells and tissues in vitro does not provide information about homeostasis or defenses involved in the responses of an intact animal to exposure by various routes, such studies can avoid some uncertainties of extrapolation from animal to human models, can provide specific, repeatable, precise measures of target-cell effects, and can help to determine their mechanisms (Pitt, 2000).

Hirvonen et al. (1997a) tested the ability of Streptomyces annulatus and S. californicus—both gram-positive bacteria—and the fungi Candida, Aspergillus, Cladosporium, and Stachybotrys to activate the mouse macrophage cell line RAW264.7. All the microorganisms were isolated from moldy houses, and no endotoxin contamination was detected in the cell suspensions. Both bacterial species substantially induced the iNOS enzyme and increased NO, TNFα, and IL-6 production in a dose-dependent manner within 24 h. Only Stachybotrys affected cell viability.

Hirvonen et al. (1997b) compared the effect of Streptomyces species on macrophages with the macrophage response produced by the gram-positive Bacillus sp. and Micrococcus luteus, which are common airborne bacteria in normal houses, and the gram-negative bacterium Pseudomonas fluorescens, a known activator of macrophages. All Streptomyces species tested were able to induce substantial amounts of TNFα and IL-6 and to induce the expression of iNOS and later NO; Bacillus sp. and Micrococcus luteus, commonly found in houses without dampness problems, did not. None of the bacteria affected cell viability, but endotoxin LPS and Pseudomonas fluorescens substantially reduced cell viability within 4 h. For Streptomyces, some factor other than NO production seemed to be required to initiate apoptosis, but the induction of proinflammatory mediators may play a role in inflammation related to exposure.

Huttenen et al. (2000) studied inflammatory responses of RAW 264.7 macrophages to three mycobacteria isolated from a moldy building: nonpathogenic Mycobacterium terrae and potentially pathogenic M. avium-complex and M. scrofulaceum. All the bacterial species tested induced time- and dose-dependent production of NO, IL-6, and TNFα, but IL-1 and IL-10 production was not detected. Reactive oxygen species (ROSs) were increased at the highest doses. The level of response differed widely across species. The nonpathogenic M. terrae was the most potent inducer, and M. avium-complex was the least potent; both pathogenic and nonpathogenic bacteria apparently activate inflammatory processes.

Hirvonen et al. (2001) exposed RAW 264.7 macrophages to Streptomyces annulatus spores isolated from a moldy building and then grown on 15 growth media to determine whether growth conditions affected a microorganism's ability to induce inflammatory mediators. After 24 h, bacteria from all growth media induced iNOS in macrophages to some extent; the amount of NO produced ranged from 4.2 to 39.2 µM, depending on the growth medium. ROSs were induced only by the highest dose of S. annulatus grown on glycerol-arginine agar. Cytokine production (IL-6 and TNFα) depended on the growth medium. Viability of the RAW 264.7 macrophages varied widely (from 11% to 96%), depending on the growth medium on which the S. annulatus was grown.

Murtoniemi et al. (2002) tested the effects of three molds (Stachybotrys chartarum, Aspergillus versicolor, and Penicillium spinulosum) and one gram-positive bacterium (Streptomyces californicus) isolated from water-damaged buildings and then grown on different wetted plasterboard cores and liners. Both liners and cores of plasterboard supported microbial growth; all species grew earlier on the core than on the liner material. Penicillium grew only on the plasterboard cores. Aspergillus and Streptomyces grown on those building materials were the most potent of the microorganisms in inducing the production of NO and IL-6 in RAW 264.7 macrophages; Stachybotrys spores did not induce NO nor IL-6 but did induce abundant TNFα production. Aspergillus also produced high concentrations of TNFα, and both Aspergillus and Stachybotrys were potently cytotoxic.

Nielsen et al. (2001) examined the cytotoxicity of 20 Stachybotrys isolates from water-damaged buildings and their ability to induce inflammatory mediators in RAW 264.7 macrophages. Eleven of the isolates produced satratoxin and were highly cytotoxic to macrophages. Isolates that produced atranone were not cytotoxic but induced inflammatory mediators (ROS, NO, IL-6, and TNFα at doses of 10⁶ spores/mL). Pure atranone B and atranone D did not elicit such a response. It should be noted that 30–40% of Stachybotrys strains isolated from buildings produce satratoxin (Jarvis et al., 1998).

Animal Experiments

Jussila et al. (2001) compared the mouse inflammatory response to a single intratracheal instillation of one of three doses of Streptomyces californicus spore isolates from the indoor air of moldy buildings with the response to 50 µg of lipopolysaccharide (LPS). Effects were assessed daily for 7 days after dosing. Cytokine concentrations were measured in the blood and bronchoalveolar lavage fluid (BALF). Histologic tests were conducted on two mice from each exposure group. S. californicus spores induced acute inflammation in mouse lungs, measured in BALF and histologically. The inflammation was still detectable 7 days after exposure. The pattern of cytokine production and the histologic effects were distinguishable from those caused by LPS. Production of the proinflammatory cytokines IL-6 and TNFα was increased in a dose-dependent manner.

Jussila and colleagues have also studied inflammatory and toxic responses to the bacteria S. californicus (Jussila et al., 2001, 2003) and Mycobacterium terrae (Jussila et al., 2002a) and the fungi Aspergillus versicolor (Jussila et al., 2002b) and Penicillium spinulosum (Jussila et al., 2002c) after intratracheal instillation in specific-pathogen-free mice. All treatments enhanced TNFα and IL-6 production in BALF after a single dose, but there were marked differences in the time course and magnitude of the response. Details of the responses are provided in Table 4-2. Except for M. terrae-treated mice, TNFα concentrations were indistinguishable from those in controls by 3 days after exposure. Both bacterial species induced inflammation at their lowest dose; the fungal spores required higher doses for induction of TNFα response. All the microorganisms increased the total number of inflammatory cells in BALF. Neutrophils were the most typical cells recruited for the acute inflammatory response, and their response peaked at 24 h; the response of macrophages peaked at 3 days, and that of lymphocytes at 7 days.

TABLE 4-2

Summary of Inflammatory and Toxic Responses to Two Bacteria and Two Fungi in Mice.

Repeated dosing with S. californicus induced mild to abundant increases in the numbers of mononuclear cells and neutrophils in the alveoli and in the bronchiolar lumen (Jussila et al., 2003). The numbers of peribronchial cells and vascular mononucleated cells also increased. Granuloma-like lesions were seen in one of three mice. Both M. terrae and S. californicus provoked systemic effects in the lymph nodes and spleen and increased TNFα and IL-6 in the blood.

Immunotoxicity

Immunotoxicity can result from immunosuppression after exposure to a xenobiotic or from immunoenhancement, autoimmunity, and allergic reactions. Immunosuppression can increase susceptibility to infectious disease and cancer through the loss of immunosurveillance cells (Corrier, 1991; Corrier and Norman 1988). It can result from decreased activity of any of the immune cells, their precursors, or other immune-related cells through inhibition of function, decrease in their population, or other dysregulation. Interpretation of data regarding immunologic end points is extremely difficult, but many mycotoxins have been found to affect alveolar macrophages in in vitro studies. Some of those studies are summarized here.

Alveolar macrophages (called pulmonary macrophages in some studies) are part of the physical defense mechanism of the respiratory system. They help to clear particulate materials, including infectious organisms, from the lower respiratory system by phagocytosis (engulfing, killing, and digesting), or by transporting them out of the respiratory system via the mucociliary escalator. When they and their particulate burden reach the oropharynx, they are swallowed. Alveolar macrophages may also transport particulate material into the lymphatic system and to the regional lymph nodes. Lung-associated lymph nodes contain antibody-forming cells that can be stimulated to form specific antibodies against antigenic material brought to them by alveolar macrophages (Haley, 1993).

Indicators of increased susceptibility to infectious disease are seen in animal field investigations in which flocks of sheep, herds of pigs, or flocks of birds fail to thrive and exhibit reduced immune response to common infectious organisms after exposure to a microbial agent (Corrier, 1991; WHO, 1990). Such responses appear at lower exposures than more overt signs of toxicity, such as vomiting, staggering, or hemorrhage (Corrier, 1991; Smith and Moss, 1985; WHO, 1990). Immunotoxic studies in animals generally examine the effects of short-term exposures. Table 4-3 lists some immunoactive mycotoxins, the fungi that produce them, their potency expressed by various measures, their mechanisms of action (if known), and their effects.

TABLE 4-3

Immunoactive Mycotoxins and Effects.

Inhibition or modulation of immune defenses results from exposure to a variety of mycotoxins. According to Pier and McLoughlin (1985), three groups of mycotoxins are predominantly associated with immunosuppressive toxicity: aflatoxins, OTA, and trichothecenes. In all those groups, inhibition of protein synthesis plays a role, although each group acts on a different site of protein formation (Corrier, 1991): aflatoxins bind to DNA and interfere with transcription of RNA from DNA (Hsieh et al., 1977), OTA inhibits the enzyme phenylalanyl-t-RNA synthetase (McLaughlin et al., 1977), and T-2 toxin (representing several other trichothecenes) prevents initiation of translation of mRNA into protein (Hsieh et al., 1977; McLaughlin et al., 1977).

Jakab et al. (1994), using a nose-only exposure chamber, examined inhalation exposure of rats and mice to nebulized AFB₁ (particle mass median diameter, 0.2 µm; time-weighted average concentration, 3.17 µg/L). An initial experiment demonstrated a clear dose-dependent decrease in Fc receptor-mediated alveolar macrophage phagocytosis 3 days after exposure for 20, 60, and 120 min. In a second experiment, alveolar macrophage phagocytosis was measured 2, 4, 7, and 13 days after exposure; a single dose of AFB₁ aerosol suppressed alveolar macrophage phagocytosis for nearly 2 weeks. In a third experiment, 250-µL suspensions of 50 and 150 µg of crystalline AFB₁ were instilled, and alveolar macrophage phagocytosis was assessed 1, 3, and 7 days after exposure by measuring the capacity of alveolar macrophages to produce TNFα elsewhere after LPS stimulation. TNFα production was suppressed in a dose-dependent manner on all days. Similar results were seen in mice into which increasing doses of AFB₁ were instilled intratracheally when alveolar macrophage phagocytosis was measured 4 days after exposure. As was seen with rats, alveolar macrophage phagocytosis was suppressed 4 days after exposure in a dose-dependent manner.

Jakab et al. (1994) also assessed systemic immune function by measuring primary splenic antibody responses to sheep red cells (SRBCs). Swiss mice immunized with SRBCs a day before or a day after AFB₁ treatment were exposed by inhalation or intratracheally to 75 µg of AFB₁. Splenic antibody response was measured 5 days after immunization by counting the antibody-forming cells. Respiratory tract exposure to AFB₁ significantly suppressed the primary antibody response to SRBCs; the effect was greater when AFB₁ treatment preceded immunization. Neither histologic nor inflammatory alterations occurred as a result of inhalation or intratracheal instillation of AFB₁. Those experiments indicate that suppression of alveolar macrophages could suppress the clearance of particles from the lung, the killing of bacteria, the suppression of tumor cells, and the modulation of inflammatory and immune processes, both through suppression of phagocytosis and through the inhibition of TNFα. Systemic immune function might also be suppressed.

T-2 toxin increased mortality from Salmonella administered orally to chickens a week after toxin exposure (Boonchuvit et al., 1975), but neither T-2 toxin nor Salmonella caused mortality by itself. Similarly, mice challenged with Mycobacterium obvis after T-2 treatment died early and in greater numbers than those not treated with this trichothecene (Otokawa, 1983). Mice treated with T-2 toxin and given intracerebrally injections of Japanese encephalitis virus (JEV) 5 days after cessation of toxin treatment died within 4 days (only one of 10 control mice died). In the early phase of JEV infection, however, the immune response does not seem to play a role in lethality, and the mechanism underlying the effect of T-2 toxin is not known (Otokawa, 1983). Infection experiments show that repeated administration of trichothecenes in animals induces more susceptibility to microbial infection (Otokawa, 1983).

Sumi et al. (1987) exposed (by diet, inhalation, and dermal exposure) 21 male Wistar germ-free rats to Aspergillus versicolor in a germ-free isolator for 2 years and compared them with 20 nonexposed controls. Histologic examination found necrosis of the liver (86% of the animals), foamy-cell granuloma of the lung (76%), fibrosis of the pancreas (52%), and nephritic lesions (57%) in the exposed animals. None of the control animals had lesions. Of the exposed rats, 62% exhibited tumors of the pleura, lung, and endocrine organs, compared with 15% of controls. Two exposed rats developed mesothelioma, and another developed squamous-cell carcinoma of the lung. Those results indicated that high-concentration exposure to A. versicolor in the absence of other microorganisms induced severe organ damage and some tumors in the rats.

In another study, Sumi et al. (1994) exposed germ-free rats to A. versicolor to elucidate the mechanism of lung damage from exposure to A. versicolor-contaminated grain dust. They exposed 21 rats for 2 years to a pure culture of A. versicolor and evaluated them 1, 2, 3, and 6 months after exposure; the results were compared with those in 21 germ-free controls. After 1 month, alveolar macrophages increased in number and became foamy macrophages as they ingested and digested mold spores. The macrophages expressed IL-1, IgA antigens, and intercellular adhesion molecules intensely bound to lymphocytes. Numerous lymphocytes infiltrated granulomatous lesions consisting of accumulated foamy macrophages and some T lymphocytes, which carried the IL-2 receptor. Granulomatous lesions extended throughout the lung, especially around bronchioles, and were present from the alveolar ducts to alveolar spaces up to 6 months after exposure. The authors concluded that macrophages may be a key effector in producing granulomas of the lung and that inhalation of A. versicolor at high concentrations may induce lung damage even in the absence of microbial infection.

Acute and chronic exposures to trichothecene mycotoxins result in depletion of lymphoid tissues, an indication of immune dysfunction, but in vitro experiments indicate that the trichothecenes have both immunosuppressive and immune-enhancing effects (Biagini, 1999). Major trichothecenes whose immunosuppressive activity has been reported are T-2 toxin, DAS, and stachybotryotoxin (Pier and McLoughlin, 1985). Their activity is associated in mice, rats, cattle, turkeys, and guinea pigs with alterations in serum proteins and immunoglobulin profiles, reduced antibody formation, thymic aplasia, reduced cell-mediated immune responses, increased delayed cutaneous hypersensitivity, and impaired bacterial clearance and acquired immunity (Pier and McLoughlin, 1985). Trichothecene immunosuppression also appears to be due to interference with the generation of suppressor cells for the delayed hypersensitivity response (Ueno, 1989) and inhibition of protein synthesis (Hughes et al., 1989). T-2 toxin reduces complement (primarily C₃) formation, diminishes serum immunoglobin (IgA and IgM, but not IgG), and diminishes antibody production (Pier and McLoughlin, 1985). Cells that depend on a high rate of protein synthesis—such as lymphoid cells, those lining the gastrointestinal tract, and hematopoietic cells—seem to be most sensitive to that effect of trichothecene exposure.

OTA inhibits protein synthesis through inhibition of phenylalanyl t-RNA synthetase (McLaughlin et al., 1977). Various effects—including necrosis of the lymph nodes, inhibition of macrophage migration, and reductions in immunoglobulin and antibody production—have been shown to result from that inhibition. The last effect seems most important, and, in contrast with the effects of aflatoxin, there does not seem to be inhibition of complement or cell-mediated immunity (Pier and McLoughlin, 1985). AFB₁ has been shown experimentally to produce thymic aplasia, to reduce T-cell function and number, to diminish antibody response, to suppress phagocytic activity, and to reduce complement (Pestka and Bondy, 1990).

Richard and Thurston (1975) tested the effect of a mixture of aflatoxin (AFB₁, AFB₂, AFG₁, and AFG₂) exposures on phagocytosis of Aspergillus fumigatus spores by rabbit alveolar macrophages. Rabbits were exposed orally to daily doses of 0.03, 0.05, and 0.07 AFB₁ equivalents/mL for 2 weeks. Aspergillus fumigatus spores were mixed with serum extracted from control and treated rabbits. Rabbits were sacrificed, and macrophages from their lungs were cultured, tested for viability, and inoculated with serum containing the spores. Macrophages from rabbits given any of the doses of aflatoxin in rabbit serum had lower phagocytic activity than controls; the extent of the reduction was dose-dependent. Fresh or frozen, but not heat-treated, rabbit serum was required for any significant phagocytosis by cultured macrophages; this indicated that the reduction of phagocytosis by aflatoxin treatment could be related to a lowering of complement or some other opsonization² factor.

Cusumano et al. (1996) used monocytes isolated from healthy human volunteers to study the effects of AFB₁ (0.05, 0.1, 0.2, 0.3, 0.4, 0.5, and 1.0 pg of AFB₁/mL for 2 and 24 h) on phagocytosis, response to microbial activity, superoxide production, and intrinsic antiviral activity. The phagocytic activity of human monocytes was dose-dependent and was significantly lower than controls after 2 hours of pretreatment at 0.5 and 1 pg of AFB₁/mL. Pretreatment for 24 h reduced phagocytic activity significantly at all doses tested. Pretreatment with 1.0 and 0.5 pg/mL for 2 h significantly impaired killing of the yeast Candida albicans, and 24-h pretreatment significantly increased the degree of impairment. Production of superoxide anion and antiviral activity were not significantly changed.

Dolimpio et al. (1968) demonstrated an inhibition of mitosis in human leukocytes isolated from three healthy female volunteers after an 8-h exposure to AFB₁ (1–50 µg/mL); greater inhibition was seen after a 48-h exposure. The inhibition was time- and dose-dependent. Chromosomal aberrations—including gaps, breaks, fragments, deletions, and translocations in different chromatids—were also seen.

Pier and McLoughlin (1985) summarized the mechanisms of aflatoxin immunosuppressive actions as follows:

• Most studies suggest that aflatoxin impairs the immune response without affecting antibody formation.
• Aflatoxin suppresses complement (C₄) and interferon, nonspecific circulating substances related to resistance to infection.
• Aflatoxin suppresses macrophage phagocytosis.
• Aflatoxin causes thymic aplasia.
• Aflatoxin suppresses cell-mediated immunity, especially delayed cutaneous hypersensitivity, lymphoblastogenesis, and leukocyte migration.

Sorenson et al. (1985) studied the toxicity of patulin, which is produced by several species of Aspergillus and Penicillium and has been shown to have the capability to be mutagenic, teratogenic, and carcinogenic. The researchers looked at cell leakage, energy metabolism, and protein synthesis in alveolar macrophages. Leakage of ⁵¹Cr from alveolar macrophage, after exposure to 0.15 mM patulin was time- and concentration-dependent. ATP concentrations were markedly inhibited within 1 h at patulin concentrations of less than 0.05 mM. RNA synthesis and protein synthesis were strongly inhibited with RNA and protein synthesis ED₅₀s (effective doses for 50% inhibition at 1 h) of 0.0016 and 0.019 mM, respectively. Protein synthesis was a much more sensitive end point than RNA synthesis, cell leakage, and ATP concentrations, all of which were more sensitive than cell volume.

Sorensen and Simpson (1986) examined the toxicity of penicillic acid—a toxin similar in chemical nature, molecular size, and toxic end points to patulin that is produced by Penicillium species—for rat alveolar macrophages, using similar methods as in the previous experiment. Results were similar to those for patulin in the earlier study, except that patulin is slightly more toxic then penicillic acid.

Neurotoxic Effects

Occupants of damp and moldy buildings have sometimes reported central nervous system symptoms—such as fatigue, headache, memory loss, depression, and mood swings—that they attribute to the indoor environment. However, mycotoxin exposure of those people in their environment has not been identified and measured.

Neurotoxic effects of mycotoxins have been examined in herd animals because consumption of mold-contaminated feed has led to severe neurologic diseases, such as rye grass staggers. Mycotoxins that selectively or specifically target the nervous system have been isolated from species of fungi contaminating grain in incidents of animal toxic response. Table 4-4 lists mycotoxins whose neurotoxic effects have been studied at least in some animals, the genera and species of fungi that produce them, their potency, their mechanism of action (if known or hypothesized), and their neurotoxic end points.

TABLE 4-4

Neurotoxic Mycotoxins and Effects.

Neurotoxic mycotoxins tend to fall into three general classes: tremorgenic toxins, paralytic toxins, and toxins that interfere with neurotransmitters or receptors either centrally or at the target organ. Many of the toxins are very potent and have immediate effects on animals exposed to a single dose by various routes. Few long-term exposures have been studied, and tests that would evaluate subtle changes in function of animals have not been done. Toxins that exert their effects on the nervous system by interfering with protein, RNA, or DNA synthesis or that exert their effects on membranes have been examined only for short-term exposures. Susceptibility to such toxins varies among animal species. Pigs and sheep seem to be as susceptible as other herd animals and rodents to tremorgens (El-Banna and Leistner, 1988; Peterson and Penny, 1982). Human susceptibility is not well established.

Tremor

Tremorgenic toxins are produced predominantly by Aspergillus and Penicillium species (Ciegler et al., 1976; Land et al., 1994). The penitrem-type of mycotoxins produces a neurotoxic syndrome in animals that involves sustained tremors, limb weakness, ataxia, and convulsions (Steyn and Vleggaar, 1985). Tremorgenic toxins generally initiate measurable effects in experimental animals within minutes of exposure. Norris et al. (1980) found that penitrem A (produced primarily by P. crustosum) increased spontaneous release of the endogenous neurotransmitters glutamate, γ-aminobutyric acid (GABA), and aspartate by interfering with the neurotransmitter-release mechanisms.

After sublethal doses, animals may suffer effects for hours or days but recover completely from the effects (Knaus et al., 1994; Peterson et al., 1982). Few long-term exposures have been examined, and tests that would determine subtle changes in function have not been done. Selala et al. (1989) reported that tremorgenic mycotoxins are partial agonists of GABA. Peterson et al. (1982) showed that 5-month-old lambs were more sensitive than 15-month-old sheep, and repeated dosing did not indicate a cumulative effect of verruculogen, a tremorgenic mycotoxin produced by P. crustosum and P. simplicissimum. A. clavatus is toxic to sheep and cattle in pastures; it produces a highly lethal mycotoxicosis that involves neural degeneration and necrosis of the midbrain, medulla, and ventral horns of the spinal cord. The specific toxin involved is not yet known, but it does not seem to be patulin or any other known tremorgen (Kellerman et al., 1976).

Paralysis

Penicillium species also produce neurotoxins that induce paralysis. Citreoviridin, produced by P. citreo-viride and A. terreus, and verrucosidin, produced by P. verruculosum var. cyclopium, are examples of such toxins (Franck and Gehrken, 1980; Hodge et al., 1988; Ueno and Ueno, 1972). Those toxins produce a progressive, ascending paralysis and are thought to act at the level of the interneurons and motor neurons of the spinal cord and motor nerve cells of the medulla (Ueno, 1984b). A typical pattern of poisoning begins with paralysis of the hind legs, which is followed by a drop in body temperature and respiratory arrest (Ueno and Ueno, 1972).

The tremorgenic and nontremorgenic mycotoxins from Aspergillus and Penicillium work at a different functional level of the nervous system from mycotoxins that have more widespread targets for toxicity or work by inhibiting basic cellular functions, such as protein synthesis.

Other Effects

Ochratoxin

OTA is toxic to nephrons and is a known neurotoxicant during prenatal stages (WHO, 1990). It is produced by Aspergillus and Penicillium species.

In tissue-culture experiments, 5–50 times higher OTA concentrations were required to affect inhibitory (GABA) transmitter levels than to affect markers for neuritic outgrowth and differentiation in both brain and retinal embryonic cell cultures (Bruinink and Sidler, 1997). That indicates that the OTA teratogenic, neurotoxic end point differs from that of the tremorgenic toxins. Bruinink and Sidler (1997) also found neural cells to be more sensitive to OTA than the meningeal fibroblast cultures previously studied by Bruinink et al. (1997); this supports previous suggestions based on in vivo data (Miki et al., 1994) that neural tissues are especially sensitive to OTA. Miki and colleagues (1994) found that the neurosensory and visual cortex of the brain of mice whose dams were treated with OTA during pregnancy had reduced numbers of synapses and that there was a significant deficit in brain, but not body weight, of treated vs age-matched controls.

OTA is a chlorinated dihydroisocoumarin derivative bound to phenylalanine (Phe) through an amide bond. It is an inhibitor of protein synthesis. Previous work in yeast indicated that that might be due to competitive inhibition of the aminoacylation of Phe tRNA by phenylalanyl-tRNA synthetase, and research on rat hepatocytes indicated Phe hydroxylation (Creppy, 1995; Creppy et al., 1983); but these mechanisms do not seem to be involved in brain and retinal cell cultures (Bruinink and Sidler, 1997). The differences, however, might be a function of the various concentrations of OTA used in the several sets of experiments. Bruinink and Sidler (1997) found their effects on neurite formation at concentrations much lower than those at which Creppy and co-workers saw enzyme inhibition. Thus, OTA may affect neural cell differentiation at concentrations much lower than those at which it affects basic cell functions, such as protein synthesis.

OTA has been measured in human maternal and cord blood (Jonsyn et al., 1995a) and in breast milk (Jonsyn et al., 1995b) in Sierra Leone. The consequences of such exposure to the human nervous system, however, have not been studied (Bruinink and Sidler, 1997).

Gliotoxin

Gliotoxin is an epipolythiodioxopiperazine compound that is a potent immunomodulator (Sorenson, 1993). It has been used therapeutically as an immunosuppressive agent for transplantation of organs and tissues. Neurotoxicity has been associated with its use, and data indicate that it can directly affect astrocytes (Chang et al., 1993). Neurotoxic effects might also be indirect through its effects on the immune system. Gliotoxin is commonly produced in cultures of Aspergillus fumigatus isolated from tissues of animals that have experimental aspergillosis and in naturally infected tissues (Sorenson, 1993). It might also play a role in the etiology of the disease aspergillosis (Amitani et al., 1995).

Trichothecenes

Trichothecene mycotoxins have a tricyclic trichothane skeleton with an olefinic group at carbon atoms 9 and 10 and an epoxy group at carbon atoms 12 and 13. Macrocyclic trichothecenes have a carbon chain between carbon atoms 4 and 15 that contain an ether or an ester linkage. The 12,13-epoxide ring, the double bond between carbon atoms 8 and 9, and the presence of various free ester groups are essential to trichothecene toxicity (Bamburg and Strong, 1971). Trichothecene mycotoxins are potent inhibitors of protein, RNA, and DNA synthesis; they act by binding to ribosomes in the cells of eukaryotic organisms (McLaughlin et al., 1977; Ueno, 1980, 1984a).

Because protein synthesis is fundamental to growth and maintenance of cells, inhibition of this fundamental cellular function can have profound effects. Neurotoxic effects in laboratory animals include degeneration of nerve cells in the central nervous system, vomiting, central nervous system-mediated loss of weight and failure to thrive, anorexia, and thirst (Ueno, 1984a).

T-2 toxin, produced by Fusarium species, has been used experimentally to study the effects of this class of toxins. T-2 toxin causes neurotoxic effects, including feed refusal, neuromuscular disturbances, and vomiting due to stimulus of the chemoreceptor zone of the medulla (Matsuoka et al., 1979; Weekley et al., 1989). Acute and chronic dosing of female rats with T-2 toxin differentially altered tryptophan, tyrosine, and serotonin concentrations in the cerebellar and brainstem regions; no systemic signs of toxicosis were evident during these neurotransmitter changes (Weekley et al., 1989). The authors suggest that T-2 toxin induces a central neurochemical imbalance that causes an alteration in autonomic function, which can then contribute to the cardiotoxic effects seen with T-2 toxin.

Ueno (1977) reports that T-2 toxin and its metabolites cause depression of the central nervous system that manifests as hyporeflexia, ataxia, and prostration. Bergmann et al. (1988) indicate that T-2 toxin causes cerebral toxicity.

Deoxynevalinol (also called vomitoxin) is a trichothecene produced by Fusarium species that is thought to affect 5-hydroxytryptamine receptors in the peripheral nervous system; these receptors are especially prevalent in the gut and are thought to mediate vomiting (Rotter et al., 1996). Effects on the central nervous system may also be mediated through changes in transmitter concentrations in the vomiting center in the medulla (Prelusky, 1993).

Carcinogenesis

Some bacteria and molds found in indoor environments produce molecules that are known or thought to be carcinogenic in humans and other animals (Table 4-5), and a number of toxins produced by molds are mu tagenic or clastogenic in various species. Others are transformed to carcinogenic chemical species by host metabolism (Wang and Groopman, 1999), such as the epoxide metabolite of AFB₁ that is produced in the liver and lungs via cytochrome P-450 enzyme activity and is considered a possible human carcinogen by the International Agency for Research on Cancer (IARC, 1993). AFB₁ is produced by Aspergillus flavus and A. parasiticus. A. flavus causes a problem in agricultural grains and peanuts grown and stored in hot humid conditions, primarily in tropical and subtropical climates. Contamination with AFB₁ is associated with high rates of hepatocarcinoma in some African countries and appears to potentiate the hepatocarcinogenic properties of hepatitis B virus through its immunotoxic effects (Autrup et al., 1987; Badria et al., 1999; Bechtel, 1989; Groopman et al., 1992).

TABLE 4-5

Carcinogenic Effects of Mycotoxins.

Exposure to high concentrations of dust from silos, grain, and peanut processing has been associated with liver and lung cancer in a few case studies and epidemiologic studies (Hayes et al., 1984; Olsen et al., 1988; van Nieuwenhuize et al., 1973). Concern about AFB₁-associated grain-dust inhalation by workers led to an intratracheal instillation and inhalation nose-only study of rats exposed to AFB₁ (Zarba et al., 1992). Maximal DNA binding of AFB₁ occurred within 30 min in the livers of the animals and indicated that inhalation exposure results in genotoxic damage to the liver; lung binding of AFB₁ to DNA was not assessed. A. flavus is generally not found indoors in northern climates, although it has been isolated from soil of indoor plants. It is found indoors more frequently in warm climates. AFB₁ is activated to a carcinogenic epoxide by human lung microsomes, but the cells that contain the activation enzymes are in low concentration in the human lung compared with human liver (Kelly et al., 1997). However, A. versicolor, which produces a precursor of aflatoxin, has been shown to induce tumors in germ-free rats (Sumi et al., 1987).

According to the U.S. National Toxicology Program's 10th report on carcinogens, OTA is reasonably anticipated to be a human carcinogen on the basis of sufficient evidence of carcinogenicity in experimental animals (NTP, 2002). It is produced by A. ochraceus, A. alutaceus, and P. viridicatum, verrucosum, and cyclopium, which are fairly common contaminants of grain and other foodstuffs. Although molds producing ochratoxins are occasionally found growing indoors, no study of their potential carcinogenic role from indoor exposures has been done. IARC (1993) concluded that there was inadequate evidence of carcinogenicity of OTA in humans but noted that is implicated in high rates of Balkan endemic nephropathy.

Citrinin, produced by P. aurentiogriseum, is often found with OTA, but is less potent (Krogh, 1989, 1992). Mayura et al. (1984) have produced experimental evidence of interactions between OTA and citrinin.

Molds that produce carcinogenic mycotoxins have been found among fungal flora indoors, but few studies that have isolated the toxins or looked for biomarkers of exposure have been conducted. All the studies that have implicated inhalation exposures related to cancers were of massive exposures to grain or peanut dust that contained spores with AFB₁ at concentrations hundreds of thousands of times greater in air than those thought to be present in indoor, nonagricultural environments. Therefore, the relevance of such exposures to those due to damp indoor spaces is unknown.

FINDINGS, RECOMMENDATIONS, AND RESEARCH NEEDS

On the basis of its review of the papers, reports, and other information presented in this chapter, the committee has reached several findings and recommendations and has identified several research needs regarding the nonallergic effects of molds and bacteria found in damp indoor environments.

• Molds that can produce mycotoxins under the appropriate environmental and competitive conditions can and do grow indoors. Damp indoor spaces may also facilitate the growth of bacteria that can have toxic and inflammatory effects. Little information exists on the toxic potential of chemical releases resulting from dampness-related degradation of building materials, furniture, and the like.
• In vitro and in vivo studies have established that exposure to microbial toxins can occur via inhalation and dermal exposure and through ingestion of contaminated food. Animal studies provide information on possible target organs, the underlying mechanisms of action, and the potency of many toxins isolated from environmental samples and substrates from damp buildings. The dose required to cause adverse health effects in humans has not been determined.
• In vitro and in vivo studies have demonstrated adverse effects—including immunotoxic, neurologic, respiratory, and dermal responses—after exposure to specific toxins, bacteria, molds, or their products.
• In vitro and in vivo research on Stachybotrys chartarum suggests that effects in humans may be biologically plausible; these observations require validation from more extensive research before conclusions can be drawn.
• Information on DNA, RNA, and protein adducts resulting from interactions with toxins is available. However, research is needed to further develop techniques for detecting and quantifying mycotoxins in tissues in order to inform the question of interactions and the determination of exposures resulting in adverse effects.
• Animal studies should be initiated to evaluate the effects of long-term (chronic) exposures to mycotoxins via inhalation. Such studies should establish dose-response, lowest-observed-adverse-effect levels, and no-observed-adverse-effect levels for identified toxicologic endpoints in order to generate information for risk assessment that is not available from studies of acute, high-level exposures.

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Footnotes

1

Toxicity was characterized as a function of the amount of crude methanol-extracted solid needed to cause 50% inhibition of growth of feline fetal lung cells.

2

The coating of a particle with a substance that helps it to attach to a phagocytic leukocyte.