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Damp Indoor Spaces and Health (2004)

Chapter:4 Toxic Effects of Fungi and Bacteria

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Suggested Citation:"4 Toxic Effects of Fungi and Bacteria." Institute of Medicine. 2004. Damp Indoor Spaces and Health. Washington, DC: The National Academies Press. doi: 10.17226/11011.
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4 Toxic Effects of Fungi and Bacteria Although a great deal of attention has focused on the effects of bacteria and fungi mediated by allergic responses, these microorganisms also cause nonallergic responses. Studies of health effects associated with exposure to bacteria and fungi show that respiratory and other effects that resemble allergic responses occur in nonatopic persons. In addition, outcomes not generally associated with an allergic response—including nervous-system effects, suppression of the immune response, hemorrhage in the mucous membranes of the intestinal and respiratory tracts, rheumatoid disease, and loss of appetite—have been reported in people who work or live in build- ings that have microbial growth. This chapter discusses the available ex- perimental data on those nonallergic biologic effects. It first discusses the bioavailability of the toxic components of fungi and bacteria and the routes of exposure to them and then summarizes the results of research on various toxic effects—respiratory, immunotoxic, neurotoxic, sensory, dermal, and carcinogenic—seen in studies of microbial contaminants found indoors. It does not address possible toxic effects of nonmicrobial chemicals released under damp conditions by building components, furniture, and other items in buildings; chemical releases from such materials are discussed in Chapter 2. Except for a few studies on cancer, toxicologic studies of mycotoxins are acute or short-term studies that use high exposure concentrations to reveal immediate effects in small populations of animals. Chronic studies that use lower exposure concentrations and approximate human exposure more closely have not been done except for a small number of cancer studies. 125

126 DAMP INDOOR SPACES AND HEALTH Chapter 5 discusses human health effects and includes some case reports relevant to toxic end points. CONSIDERATIONS IN EVALUATING THE EVIDENCE Most of the information reviewed in this chapter is derived from stud- ies 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. Esti- mates of exposures of humans to spores, bacteria, microbial fragments, and dust that contains mycotoxins are inherently imprecise and imperfect; bio- markers 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 toxico- dynamics, the mechanisms that account for biologic effects, and the me- tabolism 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 regula- tory 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,

TABLE 4-1 Some Mycotoxins and the Microorganisms That Produce Them Microorganisms That Chemical Compound Produce Mycotoxins References Ergot alkaloids Claviceps purpurea, species of Sorensen, 1993; Larsen Aspergillus, Rhizopus, Penicillium et al., 2001 Substituted coumarins, for example, Several species of Aspergillus, Penicillium van Walbeek et al., 1969; aflatoxins from Aspergillus flavus, Sorensen, 1993 Aspergillus parasiticus; ochratoxins Quinones, for example, citrinins Several species of Aspergillus, Penicillium Sorenson, 1993; Malmstrom et al., 2000 Anthoquinones, for example, rugulosin Penicillium islandicum Trichothecenes (sesquiterpenes with Fusarium and Stachybotrys species Etzel, 2002 trichothecene skeleton, olefinic group at C-9, 10, epoxy at C-12, 13), for example, T-2 toxin; DON (deoxynivalenol or vomitoxin) Macrocyclic trichothecenes (having carbon Stachybotrys, Myrothecium, others Sorensen, 1993; Jarvis, 1991 chain between C-4 and 15 in ester or ether linkage, for example, satratoxins G, H; verrucarins B, J; trichoverrins A, B) Substituted furans, for example, citreoviridin Penicillium citreoviride Nishie et al., 1988 Epipolythiodioxopiperazines, for example, At least six species of Aspergillus, Waring and Beaver, 1996 gliotoxin Penicillium Lactones, lactams, for example, patulin, Penicillium, Stachybotrys Jarvis et al., 1995, 1998 stachybotrylactones, stachybotrylactams Estrogenic compounds, for example, Many species of Fusarium Betina, 1989; Kuiper- 127 zearalenone Goodman et al., 1987

128 DAMP INDOOR SPACES AND HEALTH concerns that toxins from microorganisms that grow in damp indoor envi- ronments 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. 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). Inter- species differences in susceptibility can result from differences in absorp- tion, 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. Mycotox- ins 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 inha- lation depends in part on the size of the particles formed, because their size determines where they are deposited in the respiratory tract and this deter- mines bioavailability. Figure 4-1 shows the relationship between spore di- ameter 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

TOXIC EFFECTS OF FUNGI AND BACTERIA 129 0.8 70 0.7 Lung Respiratory Area Deposition 60 Deposition Coefficient Average Spore Diameter ( µm) 0.6 Average Spore Diameter 50 (Decimal Percent) 0.5 40 0.4 30 0.3 20 0.2 0.1 10 0 0 Arthirinium Aspergillus Penicillium Cladosporium Fusarium Paecilomyces Aureobasidium Curvularia Memnoniella Botrytis Stachybotrys Ulocladium Pithomyces Alternaria Bipolaris Dreschlera Epicoccum Oidium Peronospora Stemphyllium Genus FIGURE 4-1 Spore-deposition coefficients of mold genera in indoor environments. SOURCE: Miller et al., 2001. 100 % Spores Deposited in Respirable 80 Area of Lung 60 40 20 0 0 1 2 3 4 5 6 7 8 10 Spore size (µm) FIGURE 4-2 Percentage of inhaled spores that are deposited in respirable (alveo- lar) area of lung. SOURCE: Miller et al., 2001.

130 DAMP INDOOR SPACES AND HEALTH 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. 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 ab- sorbed 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 resi- dence 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 concentra- tion of individual toxicants changes with time and location in the exposure mixture. That is particularly true for toxic compounds originating in micro- bial contaminants of indoor spaces, because growth and metabolism of microbial organisms introduce additional variables into the exposure para- digm. 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. Experimental Data Because inhalation appears to be an important route of exposure for humans, determining the bioavailability of mycotoxins after inhalation expo- sure is important for determining the relationship between damp indoor

TOXIC EFFECTS OF FUNGI AND BACTERIA 131 spaces and human health. However, compared with ingestion, relatively few animal experiments have been performed with the inhalation pathway. Some of the studies that have been conducted indicate that acute inhalation expo- sure, at least of some toxicants, is at least as toxic as exposure by intravenous injection and is more toxic than ingestion or parenteral exposure. Ueno (1984a) found that inhalation, skin, and parenteral exposure of newborn, young, and older mice to T-2 toxin—a trichothecene mycotoxin produced by Fusarium species whose LD50 (the lowest dose that kills half the animals that receive it) does not vary much across animal species (Ueno, 1980)—directly affected capillaries, increasing their permeability and lead- ing to intestinal bleeding, diarrhea, and death. However, some ingestion of the toxin might have occurred because of grooming behavior of the animals after it was deposited on their skin. The authors also noted that newborn and young animals were much more susceptible to the mycotoxin than the older mice. Marrs et al. (1986), using head-only exposures, compared the acute inhalation toxicity of T-2 toxin in guinea pigs, which tend to be sensitive to respiratory irritants, with effects of subcutaneous administration. Respira- tory rate and minute volume were measured with whole-body plethysmog- raphy. The inhaled dose was estimated by using the concentration of T-2- fluorescein-complexed aerosol collected on a filter at 1.0 L/min. The lethal concentration (LCt50), the air concentration lethal to 50% of the exposed group of animals, was determined by using a range of concentrations and exposure durations. The corresponding dose at which 50% of the exposed group dies (LD50) was estimated from the LCt50. Another group of animals received subcutaneous injections of doses of T-2 toxin ranging from 0.5 to 4.0 mg/kg. The LD50 estimated from the inhalation exposure was about twice that of the subcutaneous LD50 values, but the authors noted that only about half the inhalation dose was retained. Taking the low retention into account, the lethal dose after inhalation was similar to that by subcutane- ous injection. The types of effects on the gastrointestinal tract were similar for the two routes of exposure and are thought to be mediated systemically. The estimated LD50s were also similar to those seen by DeNicola et al. (1978) after oral dosing, and similar gastrointestinal effects have been seen in other oral-exposure studies and appear to be largely independent of route of administration (Ueno, 1984a,b). Creasia et al. (1987) exposed young adult and mature mice to T-2 toxin by inhalation for 10 min. Tremors, stilted gait, and, in some animals, prostration were observed. Animals in the highest-dose group died 5 h after exposure. After 24 h, the LCt50s were 0.08 ± 0.04 and 0.325 ± 0.010 mg/L of air for young and mature mice, respectively. The corresponding LD50s were 0.24 and 0.94 mg/kg. When those results are compared with results of other studies, inhalation exposure was about 5–10 times more potent than

132 DAMP INDOOR SPACES AND HEALTH intraperitoneal administration, which had a reported LD50 of about 4.5 mg/ kg (Bamburg, 1976; Creasia et al., 1987), and at least 10 times more potent than dermal application, which had a reported LD50 of at least 10 mg/kg (Schiefer and Hancock, 1984). Creasia et al. (1990) conducted a nose-only, acute inhalation study of the effects of a 10-min exposure to T-2 toxin in rats and guinea pigs. Respiratory-tract lesions were minimal, and lesions to organs were similar to those described after following systemic administration. LCt50s were 0.02 and 0.21 mg/L of air for rats and guinea pigs, respectively. Deposition dose was measured by extraction of toxin from sacrificed animals, and LD50 of 0.05 and 0.4 mg/kg, respectively, were estimated. In that study, inhalation exposure to T-2 was about 20 times as toxic in rats and twice as toxic in guinea pigs as in studies of intraperitoneally administered T-2 toxin (LD50, 1 mg/kg in the rat; and 1–2 mg/kg in the guinea pig. Coulombe et al. (1991) administered 3H-labeled aflatoxin B1 (AFB1) adsorbed to grain dust or in its crystalline form intratracheally to male rats and sampled blood and tissue at selected intervals for 3 weeks to determine the pharmacokinetics of this toxin. After absorption, distribution followed a two-compartment model, with an initial rapid-distribution phase fol- lowed by a slower phase. The rate of absorption from the dust-associated dose was much lower for the first 90 min and the time to peak plasma concentration was much longer (12 vs 2 h) than for the crystalline form. Clearance was identical in the two groups. At 3 h, there was a substantially greater amount of AFB1-DNA adducts in the trachea and lung of the dust group. Retention of dust-associated carcinogens in the lung is an important factor in pulmonary carcinogenesis; it presumably increases the time during which metabolically active cells of the respiratory epithelium capable of transforming procarcinogens to carcinogens are in contact with the car- cinogen. In the liver, however, the DNA binding was greater for the crystal- line group at 3 h and at 3 days. Zarba et al. (1992) found that nose-only inhalation exposure of rats to aerosolized grain dust that contained AFB1 resulted in a linear dose-response relationship (correlation coefficient, 0.96) between time of exposure and AFB1-DNA adducts for 20, 40, 60, and 120 min of exposure. Adduct formation in the lung was not determined. Dermal absorption of mycotoxins varies. When toxins in excised hu- man skin were tested, the relative penetration rate of toxins dissolved in methanol was T-2 > diacetoxyscirpenol (DAS) > satratoxin H (a tricho- thecene mycotoxin found in Stachybotrys) > AFB1 (Kemppainen et al., 1988). Systemic toxicity after dermal exposure to a mycotoxin depends on its rate of absorption, relative blood flow to skin area, and the potency of the compound and its metabolites. Of the trichothecenes studied in vivo, relative local and systemic toxicity measured by skin irritation and lethality, respectively, is T-2 > DAS ≈ verrucarin. In vitro studies are fairly consistent

TOXIC EFFECTS OF FUNGI AND BACTERIA 133 with in vivo penetration studies, although the potency of both T-2 and verrucarin is greater than that of DAS (Kemppainen et al., 1988). Kemp- painen and colleagues (1988) showed that both aflatoxins and tricho- thecenes can be absorbed through the skin. Dermal absorption is slow, but increases with the concentration of toxin, with coexposure to solvents (such as DMSO) that enhance penetration, and when the application site is oc- cluded with clothing or wraps. Joffe and Ungar (1969) showed that aflatox- ins applied to the skin of rabbits penetrated the stratum corneum and caused changes in the epidermis and dermis. Experiments in newborn, young, and adult mice (Ueno, 1984a,b) and in vitro experiments (Kemppainen et al., 1988) have demonstrated skin penetration of tricho- thecenes. Kemppainen et al. (1984) showed with 3H-T-2 toxin that T-2 toxin adsorbed onto corn dust can partition and penetrate excised human and guinea pig skin; this indicates that mycotoxin on dust is available for absorption via skin. Those studies indicate that toxins found in damp in- door spaces are bioavailable to people through inhalation and dermal expo- sure, with the more potent route of exposure depending on the compound. The extent of exposure that occurs in damp indoor spaces, however, has not been studied. 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, immuno- toxicity, neurotoxicity, sensory irritation (irritation of nerve endings of the common chemical sense), dermotoxicity, and carcinogenic effects attrib- uted 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 impor- tant in normal host defenses, but chronic or excessive release of inflamma- tory 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

134 DAMP INDOOR SPACES AND HEALTH 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 mac- rophage 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 man- ner 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 fluores- cens, 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 Streptomy- ces, 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

TOXIC EFFECTS OF FUNGI AND BACTERIA 135 macrophages to three mycobacteria isolated from a moldy building: non- pathogenic 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 Strepto- myces annulatus spores isolated from a moldy building and then grown on 15 growth media to determine whether growth conditions affected a micro- organism’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. annul- atus grown on glycerol-arginine agar. Cytokine production (IL-6 and TNFα) depended on the growth medium. Viability of the RAW 264.7 macro- phages varied widely (from 11% to 96%), depending on the growth me- dium 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 build- ing 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 Aspergil- lus 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 inflam- matory mediators in RAW 264.7 macrophages. Eleven of the isolates pro- duced 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).

136 DAMP INDOOR SPACES AND HEALTH Animal Experiments Jussila et al. (2001) compared the mouse inflammatory response to a single intratracheal instillation of one of three doses of Streptomyces cali- fornicus 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 con- ducted on two mice from each exposure group. S. californicus spores in- duced acute inflammation in mouse lungs, measured in BALF and histologi- cally. The inflammation was still detectable 7 days after exposure. The pattern of cytokine production and the histologic effects were distinguish- able from those caused by LPS. Production of the proinflammatory cyto- kines IL-6 and TNFα was increased in a dose-dependent manner. Jussila and colleagues have also studied inflammatory and toxic re- sponses to the bacteria S. californicus (Jussila et al., 2001, 2003) and Myco- bacterium 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 inflamma- tion at their lowest dose; the fungal spores required higher doses for induc- tion 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 lympho- cytes at 7 days. 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 sys- temic effects in the lymph nodes and spleen and increased TNFα and IL-6 in the blood. Respiratory Effects Microorganisms and their toxins can lead to effects on the tissues and cells of the respiratory system. Some of the effects might be mediated by effects on the immune system.

TABLE 4-2 Summary of Inflammatory and Toxic Responses to Two Bacteria and Two Fungi in Mice TNFα IL-6 Response Response (Lowest Dose (Lowest Dose Increased Albumin/- to Cause to Cause iNOS-NO Inflammatory LDH Microorganism Increase) Increase) Response Cells in BALF Response Reference Streptomyces Intense and (2 × 107 Induced Strongest: 6- Increased Jussila et al., californicus rapid spores) iNOS at fold increase at within 6 h, 2001 (1 × 10 8 spores) 2 × 107 2 × 107 spores continuing spores acute for 24 h; within 24 h increases in LDH Mycobacterium 83% of that to (1 × 108 No not Second Increased Jussila et al., terrae S. californicus; spores) increased strongest, but within 24 h; 2002a biphasic; until 7 biphasic peaked at intense acute days after 14 days; acute phase followed exposure; increases in by sustained iNOS LDH; strongest phase that lasted detectable and longest more than 14 for up to dose-dependent days (1 × 108 28 days response, lasting spores) length of experiment (continued on next page) 137

TABLE 4-2 continued 138 TNFα IL-6 Response Response (Lowest Dose (Lowest Dose Increased Albumin/- to Cause to Cause iNOS-NO Inflammatory LDH Microorganism Increase) Increase) Response Cells in BALF Response Reference (28 days) Aspergillus Rapid; peaked Massive at NA Dose-dependent Slower Jussila et al., versicolor after 24 h at 7- 1 × 10 8 spores increase starting response; 2002b fold increase (1 × 106 at 1 × 106 spores acute (1 × 10 6 spores) spores) increases in LDH Penicillium 8-fold increase Highest level; NA Minor cell Mildest Jussila et al., spinulosum within 6 h; 20-fold response even response; 2002c disappeared increase; at 5 × 106 spores no changes in more rapidly peaked later cytotoxicity than other than response responses to TNFα; was (5 × 10 6 spores) at control level 24 h after exposure (5 × 10 6 spores)

TOXIC EFFECTS OF FUNGI AND BACTERIA 139 Animals and Animal Cells Pang et al. (1987) studied the effects of a single nebulized dose of T-2 toxin at 9 mg/kg on lung tissues of young pigs (9–11 weeks old). Analyses indicated that 1.8–2.7 mg/kg was retained. Vomiting, cyanosis, anorexia, lethargy, prostration, and death occurred. Those effects are similar to those seen in pigs treated intravenously with the LD50 of T-2 toxin, 1.2 mg/kg (Lorenzana et al., 1985a,b). Pang et al. (1987) observed pulmonary and systemic immunologic and morphologic changes of the lung and other organs. Pigs were sacrificed 1, 3, and 7 days after dosing. Morphologic examination of the lungs showed small, dark red foci 2–3 mm in diameter scattered throughout the lobes. Hemorrhages of the gastrointestinal mu- cous membrane, subendocardial tissue, and subpericardial tissue were also seen. Two pigs that died after 8 h had mild to moderate patchy acute interstitial pneumonia characterized by thickening of the lung septa due to congestion and infiltration of neutrophils and macrophages. There was marked reduction in alveolar macrophage phagocytosis and mitogen- induced blastogenic responses of pulmonary, but not peripheral, lym- phocytes at 8, 24, and 72 h, but not 7 days after exposure. Thus, acute exposure to T-2 toxin resulted in mild pulmonary injury and transient impairment of pulmonary immunity (Pang et al., 1987). Nikulin et al. (1996) isolated spores from two strains of Stachybotrys atra, one more toxic1 and the other less so, from houses with moisture problems. Satratoxins G and H were present in the more toxic strains, and small amounts of stachybotrylactone and stachybotrylactam were found in both strains. A suspension of 106 spores of each strain was injected intrana- sally into a group of four 5-week-old mice. One mouse exposed to the toxic strain died 10 h after dosing, one was moribund at 24 h, and the other two survived the 3-day duration of the experiment. When observed histologi- cally, all treated mice developed inflammatory lung lesions, but the severity and extent of the lesions differed between the two groups. Spores of the more toxic strain induced severe intra-alveolar and interstitial inflamma- tion, and hemorrhagic exudate was found in the alveolar lumina. There was focal aggregation of inflammatory cells (mainly neutrophils and macro- phages), especially in the peribronchiolar area. Neutrophilic granulocytes and macrophages containing fungal spores were found in the lung paren- chyma. Some lymphocytes were found in the interstitium and necrotic changes were seen. Lungs of mice exposed to the less toxic strain of S. atra had much milder inflammatory responses, and no necrotic changes were seen. It is interesting that in this study, in which exposure was to the spores 1Toxicity 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.

140 DAMP INDOOR SPACES AND HEALTH of S. atra, all exposed mice showed pathologic changes; that was not the case in studies in which exposure was to purified T-2 toxins (Creasia et al., 1987, 1990). Nikulin et al. (1997) examined the effects of intranasal exposure of mice to S. atra spores (103 and 105) twice a week over a 3-week period, using strains and methods similar to those in their earlier work (Nikulin et al., 1996). Five groups of 10 mice (five males and five females) were treated. Mice were evaluated for weight change and blood characteristics over a 3- week period. The sixth and last administration was followed by blood- antibody measurements and hematologic and histologic studies of lung tissue. The severity of changes in lung tissue depended on the concentration and the toxic potency of spores. Treatment with suspensions of 105 spores of the more toxic strain caused severe inflammatory changes, with hemor- rhagic exudates present in alveolar lumina. Treatment with 103 spores of the same strain produced similar but milder changes. Much milder inflam- matory changes occurred in the lungs of mice treated with 105 spores of the less toxic strain, and no inflammatory changes were seen in mice treated with 103 spores of the less toxic strain. In contrast with the earlier acute- exposure testing (Nikulin et al., 1996), necrosis was not found in the lungs of any mice treated repeatedly. The authors attributed that difference to a lower concentration of satratoxins in the spores used in the multidose experiment. Antibodies against S. atra were not detected in mice exposed to S. atra spores via inhalation, but a separate group of mice intraperitoneally immunized with spores developed IgG antibodies against S. atra, as mea- sured with enzyme-linked immunosorbent analysis. Mason et al. (1998) examined the effect of Stachybotrys chartarum conidia and isosatratoxin-F, compared to the negative control fungus Cla- dosporium cladosporioides, on surfactant production in cultures of undif- ferentiated fetal type II alveolar cells from rabbit lung, and their effects following intratracheal instillation in mice. All concentrations of conidia tested (103, 104, 105, 106 conidia/mL) and isosatratoxin-F concentrations of 10–9-10–4 M decreased surfactant production, as measured by incorpora- tion of [3H]-choline into surfactant, within 24 h. In mice treated with 50 mg of 107 conidia/mL of S. chartarum or Cladosporium cladosporioides or with 50 µL 10–7 M isosatratoxin-F, phospholipid concentrations of the four primary subfractions of surfactant (P10, P60, P100, and S100) measured with lung lavage were changed in a concentration- and time-dependent manner. P10 phospholipid, which is responsible for the surface-tension- lowering properties of the lipid monolayer of alveolar cells, was signifi- cantly increased in lungs 12 and 24 h after exposure. S. chartarum-treated animals had significantly decreased P60 phospholipid and significantly in- creased P100 phospholipid 48 h after exposure. In mice, exposure to the toxin resulted in significant increases in P10 phospholipid 12 and 24 h after

TOXIC EFFECTS OF FUNGI AND BACTERIA 141 exposure and in P60 phospholipid 24 and 72 h after exposure. Toxin exposure also significantly decreased S100 phospholipid 24 h and increased P100 phospholipid 72 h after exposure in mice. In C. cladosporoides- treated mice, P60 was decreased 24 h after exposure, but no other changes in phospholipids were seen. Thus, mouse surfactant homeostasis can be disrupted by exposure to toxic S. chartarum conidia and isosatratoxin-F but not by exposure to C. cladosporoides conidia. Such disturbances might lead to disruption of clearance mechanisms and result in increased suscep- tibility to inhaled infectious organisms, but the exact meaning of the distur- bances has not yet been explained. Rao et al. (2000a) instilled 9.6 × 106 S. chartarum spores intratracheally into rats and then performed bronchial alveolar lavage to look at biochemi- cal indicators of injury (albumin, myeloperoxidase [MPO], lactate dehy- drogenase [LDH], and hemoglobin) and leukocyte differentials. They ob- served severe inflammatory changes and interstitial inflammation with hemorrhagic exudate. Exposed animals lost 10% of their body weight within 24 h. The highest inflammatory-cell and polymorphonucleocyte (PMN) count occurred 24 h after exposure. Albumin and LDH concentra- tions were also significantly increased at that time. Hemoglobin concentra- tions were different from controls at 72 h. Because S. chartarum is not known to cause an immunoglobulin G reaction in rodents or to infect mammalian lungs, the observed injury was thought to be caused by chemi- cal constituents of the spores rather than allergy or infection. Rao et al. (2000b) extracted spores of a toxic strain of S. chartarum with methanol to reduce toxicity, instilled the spores intratracheally into 10-week-old male rats, and then analyzed their BALF for total leukocytes, differential counts of macrophages, PMNs, eosinophils, and lymphocytes 24 h after treatment. Supernatant fluid was analyzed for LDH, MPO, and albumin with spectroscopy. Results were compared with those in rats treated with unextracted spores and with saline. About 0.5 mL of a saline- suspended concentration of 2 × 106, 4 × 106, 1 × 107, and 2 × 107 spores/mL saline was instilled. Physiologic effects of acute pulmonary exposure were examined by measuring body weight, LDH, hemoglobin, blood albumin, and leukocyte, macrophage, lymphocyte, and eosinophil counts. Body weight was decreased in rats treated with non-extracted spores (up to 13% loss of body weight with no loss in saline controls) in the 24 h after expo- sure. Increased LDH (resulting from cytotoxicity and death), hemoglobin (resulting from erythrocyte infiltration from pulmonary capillary beds), and albumin, a possible early indicator of inflammation, were linearly dose- dependent. The same dose-dependent increases were not seen in methanol- extracted or saline controls. Leukocyte counts were increased, and PMNs were the major contributor to the increases. Total macrophages, lympho- cytes, and eosinophils did not increase with increasing instillate concentra-

142 DAMP INDOOR SPACES AND HEALTH tions. The authors state that 24 h might not have been enough time so see a rise in macrophages in that they saw such a rise in other rats at 72 h (unpublished data). No assessment of toxin identity or concentration was carried out. Yike et al. (2002a) developed a model technique for studying pulmo- nary toxicity in infant rats. They studied the effects of S. chartarum spores containing mycotoxins on survival (LD50), growth, lung histopathology, BALF characteristics, and pulmonary function of rat pups treated when 4 days old and observed and weighed for 14 days. Intratracheal instillation of high doses of S. chartarum spores—4–8 × 105 spores/g of body weight—led to macroscopically detected hemorrhage that was frequently fatal; 73% and 83% of animals treated with 4 × 105 and 8 × 105 spores/g, respectively, died; the LD50 was 2.7 × 105 spores/g. Conidia were present in the alveoli and distal airways of a sample of 75 treated rat pups; the number of spores was greatest 4 days after treatment, at which time they were localized in the macrophages. Acute interstitial or intra-alveolar hemorrhage was observed in 62% of treated animals vs 27% of controls, which received phosphate- buffered saline (PBS); control pups treated with ethanol-extracted spores showed only minimal incidental hemorrhage. The degree of hemorrhage was dose-dependent. Hemoglobin, an indicator of acute alveolar bleeding, was significantly higher in the BALF of treated pups (2.46 ± 0.33 mg/mL of epithelial lining fluid) than PBS pups (1.22 ± 0.17 mg/mL) and pups treated with extracted spores (1.28 ± 0.16 mg/mL). Proinflammatory cytokines and inflammatory cells in lungs were significantly higher after 3 days in pups that received 1 × 105 spores/g than in PBS and ethanol-extracted-spore controls. Those indicators of acute inflammation resolved 8 days after spore instillation. Respiration, measured with whole-body plethysmogra- phy, showed statistically significant apnea of more than 3-sec duration in treated pups (28% of exposed pups vs 0% of controls). Minute volume was increased 4 days after treatment, probably because of an increase in tidal volume. Enhanced pause (a noninvasive measure of airway resistance) was also increased. Tidal volume remained elevated at day 7, when pups developed decreased respiratory rate. The experiment of Yike et al. (2002a) differs from those of Nikulin et al. (1996), Nikulin et al. (1997), and Rao et al. (2000a,b) in that trichothecene toxicity was quantified (in toxin equiva- lents), but total toxin content (that is, phenylspirodrimanes, stachytoxins, cyclosporin, and unknown toxins found in some Cleveland strains) was not characterized. The LD50 of 2.7 × 105 spores/g (270 ng of satratoxin G per gram = 0.27 mg/kg) is similar to the values reported for T-2 toxin in other animals: 0.9 mg/kg in adult rats, intravenously; 0.8 mg/kg in monkeys, intramuscularly; 5.2 mg/kg in mice, intravenously (Wannemacher et al., 1991); and 5.2 mg/kg in mice, intraperitoneally (Ueno, 1989).

TOXIC EFFECTS OF FUNGI AND BACTERIA 143 Yike et al. (2003), using the same infant rat model technique described above, observed that the conidia of S. chartarum could germinate in the lungs of infant rats and form hyphae but could not establish an effective infection even in very young rat pups. Germination was observed frequently in the lungs of 4-day-old pups but rarely in 14-day-old pups. However, acute neutrophilic inflammation and intense interstitial pneumonia with poorly formed granulomas observed 3 days after exposure were associated with fungal hyphae and conidia. In 4-day-old pups, pulmonary inflamma- tion with hemorrhagic exudates resulting in about 15% mortality was observed compared with 0% mortality in controls that received PBS. In related studies that used the Yike et al. technique in juvenile mice, Rand et al. (2002) found that a single intratracheal injection of S. chartarum spores or toxins produced marked ultrastructural changes in alveolar type II cells. Both animals that received S. chartarum spores and animals treated with isosatratoxin-F demonstrated condensed mitochondria with separated cristae, scattered chromatin and poorly defined nucleoli, cytoplasmic rar- efaction, and distended lamellar bodies with irregularly arranged lamellae 48 h after treatment. Point-count stereologic analysis revealed a significant increase (p = 0.05) in lamellar body volume density with both treatments. Rand et al. (2003) compared juvenile mice treated with 50 mL of 1.4 × 106 S. chartarum spores/mL saline toxin at ≥ 35 ng/kg of body weight, mice treated with 50 mL of 1.4 × 106 Cladosporium cladosporioides spores/mL saline, and mice that received 50 mL of saline solution. Treatment with fungal spores of either species resulted in granuloma formation at the sites of spore impaction, but some lung tissue treated with S. chartarum spores exhibited erythrocyte accumulation in the alveolar air space, dilated capil- laries engorged with erythrocytes, and hemosiderin accumulation at spore impaction sites. Immunohistochemistry of the granulomas revealed reduced collagen IV distribution in the mice treated with S. chartarum but not C. cladosporioides. Quantitative analysis of pooled S. chartarum and C. clado- sporioides spore-impacted lungs revealed significant depression of alveolar air space in animals treated with either S. chartarum and C. cladosporioides relative to that in untreated controls. Significant (p < 0.05) alveolar accu- mulation of erythrocytes was observed: from 1.24 ± 1.4% in untreated animals to 3.44 ± 1.5% in the pooled S. chartarum mice. It increased sig- nificantly over time (p < 0.001) from 2.14 ± 1.7% 12 h, to 5.54 ± 1.5% 72 h, and remained elevated at 4.94 ± 1.4% 96 h after treatment. Treat- ment with S. chartarum spores elicited tissue responses significantly differ- ent from those associated with pure trichothecene toxin or with a non- toxigenic fungus. Gregory et al. (2004) used immunocytochemistry to evaluate the dis- tribution of the trichothecene satratoxin G in spores and mycelia of

144 DAMP INDOOR SPACES AND HEALTH S. chartarum in culture and in the lung tissues of intratracheally exposed mice. Antibodies prepared in rabbits to react against satratoxin G isolated from Cleveland strain 58–17 reacted more to spores than to mycelia; that indicated a higher toxin concentration in the spores. Mice then received 3,000 spores/g of body weight by intratracheal instillation, and the distribu- tion of immune-labeled satratoxin G in tissues was determined. The toxin was observed predominantly in alveolar macrophages, but it was also found in alveolar type II cells; this finding supported other studies that demon- strated that these cells are sensitive to S. chartarum spores, isosatratoxin, and satratoxin G exposure, all of which affect surfactant production and compo- sition in BALF (Mason et al., 1998, 2001; Sumarah et al., 1999), regulation and synthesis of pulmonary surfactant (McCrae et al., 2002), and alveolar type II cell microanatomy (Rand et al., 2002, 2003) and function (Mason et al., 1998, 2001). On the basis of immunochemistry, Gregory et al. (2003) described the localization of stachylysin (which causes lysis of red blood cells in vitro) in Stachybotrys chartarum spores and in rat and mice lungs. Stachylysin label- ing was greater around 58-06 Cleveland strain spores than the 58-17 strain 72 h after exposure. Granulomatous lesions formed in rat and mouse lungs that contained spores labeled lightly for stachylysin after 24 h and more heavily after 72 h; production of the lesions may be a relatively slow pro- cess. The highest stachylysin concentration was found in the inner walls of spores and near the spores, suggesting diffusion of the stachylysin out of the spores. Stachylysin was also localized in alveolar macrophage cytoplasm and mitochondria and in phagolysosomes. The latter suggests that phago- lysosomes might be involved in the inactivation and clearance of stachylysin. Satratoxin G was localized in lysosomes, nuclear membranes, heterochro- matin, and rough endoplasmic reticulum (RER) of alveolar macrophages (Gregory et al., 2004). Alveolar type II cells showed modest labeling of nuclear heterochromatin and RER. Flemming et al. (2004) investigated dose-response relationships (30, 300, and 3,000 spores/g of body weight) and time relationships (3, 6, 24, 48, and 96 h after intratracheal instillation) in mice exposed to macro- cyclic trichothecene-producing (JS 58-17) and atranone-producing (JS 58- 06) S. chartarum strains, comparing them with results of exposure to C. cladosporioides spores. BALF total protein, albumin, proinflammatory cytokine (IL-1β, IL-6, and TNF-α), and LDH concentrations were signifi- cantly (p < 0.05) different between fungal species (S. chartarum vs C. cladosporioides), strains (58-17 vs 58-06), spore doses, and times. Mice exposed to C. cladosporioides or S. chartarum spores showed no clinical signs of illness or respiratory distress. Total protein in BALF was signifi- cantly (p ≤ 0.001) higher after high-dose exposure to S. chartarum strain 58-17 than after all other treatments. Albumin concentrations were signifi-

TOXIC EFFECTS OF FUNGI AND BACTERIA 145 cantly higher in mouse lungs exposed to high-dose S. chartarum strain 58- 06 (p ≤ 0.001), and medium-dose (p ≤ 0.01) and high-dose (p ≤ 0.001) S. chartarum strain 58-17 than after all other treatments. The changes were similar and dose-dependent. The majority of the increased protein was albumin. The NOAEL for exposure to spores of S. chartarum strains JS 58- 17 and JS 58-06 was less than 30 spores/g of body weight; for C. clado- sporioides, it was over 300 spores/g of body weight. Although after moder- ate and high doses of S. chartarum strains the BALF composition reflected differences in strain toxicity, the BALF composition after treatment with either strain at the lowest dose was similar; spore-sequestered factors com- mon to both strains, rather than strain-dependent toxins, might be contrib- uting to lung disease. An important finding was that low doses of the two S. chartarum strains (30 spores/g of body weight) still precipitated responses that were significantly higher than those associated with C. cladosporioides or saline exposures even though there was no apparent inflammation re- sponse in mice to the two S. chartarum strains. The concentration of mac- rocyclic trichothecenes in the 30-spore/g exposure of S. chartarum strain JS 58-17 was less than that associated with the NOAEL in in vitro expo- sures (Sorenson et al., 1987) and may be associated with high concentra- tions of proteases (stachyrase A) identified by Yike et al. (2002b). Joki et al. (1993) examined the effect of volatile metabolites of mold (Trichoderma viride) and bacteria (two strains of Actinomycetes) isolated from moldy houses and Penicillium from a dry surface in a nonproblem house on the ciliary beat frequency (CBF) of guinea pig tracheal-tissue explants. The volatile metabolites of the two mold strains and the bacterial strains increased CBF significantly over negative controls (Actinomycetes, 19%; Penicillium, 25%; Trichoderma viride, 30%) after various times. The authors point out that several inflammatory mediators (bradykinin, hista- mine, and leukotriene D4) increase CBF but that the physiologic meaning of this effect is unexplained. Di Paolo and co-workers (1993) report a case of acute renal failure (acute tubular necrosis) in a woman farm worker suspected to have inhaled large amounts of ochratoxin A (OTA) because Aspergillus ochraceous was found growing on wheat dust in a closed granary and OTA was identified in the wheat. When the worker was examined clinically, she reported that dust was irritating to her lungs; but pulmonary tissues showed only modest effusion 5 days after exposure. Grain dust from the granary was placed in the bottom of a closed chamber that was ventilated with a continuous current of air passed through the wheat. Animals (four rabbits and four guinea pigs) were exposed in the chamber for 8 h to assess toxicity. One rabbit died after 16 h of exposure, one guinea pig after 24 h, and one rabbit after 34 h. An autopsy of the guinea pig revealed renal tubular necrosis. The rest of the animals were sacrificed and examined 5 days after exposure. All

146 DAMP INDOOR SPACES AND HEALTH the rabbits had fatty liver degeneration and renal tubular necrosis; one rabbit had pulmonary edema. One guinea pig had tubular necrosis, but no anomalies were found in the other three guinea pigs. No quantification of animal exposure to spores or toxins was attempted. Wilson et al. (1990) examined the action of AFB1 on cultured airway epithelial cells explanted from species with abundant (rabbit and hamster) and scarce (rat and monkey) distributions of smooth endoplasmic reticu- lum (SER) in nonciliated tracheal cells. AFB1 is metabolized by cytochrome P-450 enzymes associated with SER to compounds that are mutagenic and are capable of binding nuclear DNA, as measured by DNA-adduct for- mation. DNA binding was greatest in rabbit tissue, followed by hamster, monkey, and rat tissue. A plateau of adduct formation was reached in tissues from all species after 12 h in culture. Degenerative changes in the structure of explants, as seen with electron microscopy, were greatest in rabbit and hamster tissue. In rabbit and hamster tissue, binding of AFB1 and its metabolites—as determined by autoradiography—was greater in nonciliated secretory cells than in ciliated cells, especially in necrotic cells. In rat, tissue binding was evenly distributed between ciliated and nonciliated cells. Population densities of cells, as measured by quantitative microscopy, indicated that the nonciliated secretory cells were the target of AFB1. Humans and Human Cells Pulmonary Hemorrhage in Infants Chapter 5 details information on and reviews medical and epidemio- logic studies of the possible role of S. chartarum exposure in a cluster of cases of pulmonary hemorrhage in infants in Cleveland. This has been the subject of a great deal of attention in the scientific community and by the general public. Relevant toxicologic studies are addressed below. Jarvis and colleagues (1998) examined samples of Stachybotrys chart- arum (16 isolates) and Memnoniella echinata (a fungus closely related to S. chartarum; 2 isolates) from the air and surfaces of homes of cases (10 homes) and controls (29 homes) to determine whether these molds isolated in the Cleveland investigation were producing mycotoxins. Isolates were grown in the laboratory, and extracts were tested for cytotoxicity and for specific toxins. The cytotoxicity test was performed in a feline fetal lung cell culture using inhibition of cell proliferation. Cytotoxicity was assayed on crude extracts and on fractions of them. The majority of the cytotoxicity occurred in the fractions that contained most of the macrocyclic and tricho- verroid trichothecenes from S. chartarum, consisting of satratoxins (macro- cyclic trichothecenes) and roridin L-2 and trichoverrol B (trichoverroid trichothecenes). Toxins normally produced by S. chartarum, such as tricho-

TOXIC EFFECTS OF FUNGI AND BACTERIA 147 dermol and verrucarol and their acetates, were not capable of being de- tected with the analytic method used (high-performance liquid chromatog- raphy with ultraviolet detection). Phenylspirodrimanes, which are immune suppressants, were found in all S. chartarum and M. echinata cultures. No apparent correlation of toxicity between isolates originating from case and control homes was seen. Of the isolates, three (of nine) from case and three (of eleven) from control homes were the most toxic, and three belonging to each group were not toxic. One of the isolates of S. chartarum from a case home produced a significantly greater amount of satratoxin F than would be expected from a small culture; thus, there may be significant variation in toxin production among isolates. Although some macrocyclic trichothecenes are somewhat lipophilic, Sorenson and co-workers (1996) reported that, when extracted with a water wash, they were nearly as toxic as those extracted with methanol. Jarvis and colleagues (1998) found that about 50% of the total tricho- thecenes produced by S. chartarum was found in a water extract. The authors state that the trichothecenes are exported to the fungal-spore sur- face, where they become water-soluble by being embedded in water-soluble surface polysaccharides. Such toxins might be readily released into the aqueous microenvironment of the lung surface and diffuse to the epithe- lium, gaining access to capillaries. As previously discussed, trichothecenes cause capillaries to become leaky, and are associated with hemorrhage in tissues (Jarvis, 1995; Ueno, 1984a,b). Alveoli provide ready access to capil- laries, which may be a primary target for these toxins. Cultures of the two isolates of M. echinata from one sample from a Cleveland home did not produce macrocyclic trichothecenes but did dem- onstrate toxicity similar to that of some isolates of S. chartarum. The Cleveland isolates of M. echinata were moderately cytotoxic and produced the simple trichothecenes trichodermol and trichodermin and substantial amounts of the antifungal agent griseofulvin. M. echinata isolates also pro- duced phenylspirodrimanes (Jarvis et al., 1998). Vesper et al. (1999) explored the toxicity of strains of S. chartarum isolated from eight case and eight control homes from the Cleveland out- break and 12 strains from diverse geographic locations other than Cleve- land. The goal of their study was to determine whether the effects of strains from the Cleveland case homes were different from those of strains from control homes and strains isolated elsewhere. The strains were grown on wet wallboard for 8 weeks, and conidia were then subcultured onto sheep’s blood agar at 37°C and 23°C. Cultures were examined weekly for evidence of hemolysis. Five Cleveland strains, all from case homes, showed hemoly- sis at 37°C, and three non-Cleveland strains consistently demonstrated hemolytic activity throughout the 8-week test period. All strains were hemolytic by the end of week 5 at 37° C. None was consistently hemolytic

148 DAMP INDOOR SPACES AND HEALTH at the lower temperature. All 28 strains of S. chartarum showed some toxicity, as measured by inhibition of protein synthesis. Five of the Cleve- land strains and two of the non-Cleveland strains were highly toxic, with effects seen above 90 µg of T-2 toxin equivalents per gram wet weight of conidia; one Cleveland strain and three non-Cleveland strains had interme- diate toxicity; and the 17 other strains were consistently less toxic than 20 µg of T-2 toxin equivalents per gram wet weight of conidia. Of the 28 strains examined, only three were both highly toxic and consistently hemo- lytic; all three came from Cleveland homes where infants with bleeding lived. Two of those three strains were significantly different from other highly toxic strains on the basis of random amplified polymorphic DNA (RAPD) analysis. Although the S. chartarum strains from Cleveland homes were not more toxic than strains from other locations, the results from this study suggest that a combination of toxicity and hemolytic capability may be characteristic of the Cleveland strains, and this raises the possibility that a combination of toxins and hemolysins induced pulmonary bleeding in the infants. Identification of strains that produce this combination of factors may be possible through RAPD analysis. A strain of S. chartarum isolated from the lung of a child in Texas who had pulmonary bleeding (designated the Houston strain) was studied for hemolytic activity, siderophore production, and relation to five case- and five control-home strains from the Cleveland outbreak (Vesper et al., 2000a). Hemolysin was produced more consistently and in larger amounts in the case strains from Cleveland and in the Houston strain than in most control strains, so it might play a role in pulmonary bleeding. The case strains and the Houston strain also produced more hydroxymate-type siderophores than control strains; this suggests that they may be better able to extract iron from host cells. One control strain, however, was similar to the case strains and the Houston strain in hemolysin production. Vesper et al. (2001) characterized hemolysin isolated from S. chartarum. The hemolysin has been designated as stachylysin, a β-hemolysin. β-Hemol- ysins are produced by many bacteria and by Candida albicans and Aspergil- lus fumigatus and are associated with the virulence of these fungi. Kordula et al. (2002) isolated an enzyme, named stachyrase A, from S. chartarum isolated from the lung of a child with pulmonary hemorrhage. The enzyme is a chymotrypsin-like serine proteinase that cleaves protease inhibitors, peptides, and collagen in the lung. It is possible that the enzyme could provide mycotoxins greater access to capillaries by removing epithe- lial barriers, but whether that occurred in this case has not been further investigated. The magnitude of exposure to microbial and other agents, including Stachybotrys chartarum, in the Cleveland cluster and other clusters in which S. chartarum is thought to be a factor is not known. Some 30–40% of

TOXIC EFFECTS OF FUNGI AND BACTERIA 149 strains of S. chartarum isolated from the Cleveland cases contained macro- cyclic trichothecenes associated with hemorrhage via inhalation at high exposures in animal experiments (Jarvis et al., 1998). Phenylsirodrimanes were found in all strains while atranones were found in 60-70% of the strains, and some strains produced hemolysins and enzymes that attack collagen (Kordula et al., 2002; Vesper et al., 2000a, 2001). The body of research on S. chartarum, especially the more recent stud- ies (Andersen et al., 2002; Flemming et al., 2004; Gregory et al., 2003, 2004; Jarvis et al., 1998; Rand et al., 2002, 2003; Rao et al., 2000a,b; Vesper and Vesper, 2002; Vesper et al., 1999, 2000a,b, 2001; Yike et al., 2002a,b), provides a biologically plausible mechanism by which at least some strains of this mold could affect the lungs of young animals. Other potentially toxic molds were isolated in the Cleveland case, but their toxic potency, degree of exposure, and interactions with toxins produced by S. chartarum and the closely related fungal species Memnoniella echinata (also isolated in the Cleveland case) have not been investigated. No other cluster of similar size pulmonary hemorrhage in infants has been seen since the Cleveland outbreak, although case reports of pulmonary bleeding in infants in whose lungs or environment S. chartarum was found to be present have been published (CDC, 1997; Dearborn et al., 2002; Elidemir et al., 1999; Flappan et al., 1999; Knapp et al., 1999; Novotny and Dixit, 2000; Tripi et al., 2000; Weiss and Chidekel, 2002). The role of toxigenic fungal exposure in such cases has yet to be determined. Other Effects The effects of mycotoxin-associated fungal spores (152 isolates) from the air of damp domestic environments (503 dwellings) in Scotland were tested in a human embryonic diploid fibroblast lung cell line (MCR-5) (Lewis et al., 1994). At least 37% of the isolates, primarily those of Penicil- lium species, demonstrated cell toxicity when assayed. Of the molds pro- ducing mycotoxins, the penicillia were most numerous, accounting for 81.6% of the identified isolates. Of the penicillia identified to the species level, P. viridicatum, P. expansum, and several strains of P. chrysogenum exhibited the greatest measured toxicity in water-extract trials. Two toxic species of Aspergillus fumigatus (an opportunistic pathogen capable of invading the lung) were isolated. Clear variations in cytotoxicity were observed when two additional human cell lines were used: Chang liver cells and Detroit 98 normal human sternal bone marrow cells. Strains of P. expansum, P. chrysogenum, and P. glabrum collected from dwellings showed no toxicity with the MRC-5 cell line but did with Chang liver cell culture. In addition, a P. aurentiogriseum extract resulted in nearly 3 times the mortality of Chang liver cells than of MRC-5 cells. A penicillic acid-

150 DAMP INDOOR SPACES AND HEALTH producing strain of P. aurentiogriseum was more lethal to Detroit 98 cells than to Chang liver cells but showed little toxicity to MRC-5 cells. An additional 23 extracts showed significant toxicity, relative to vehicle con- trol, when dissolved in DMSO. Amitani et al. (1995) demonstrated in vitro that Aspergillus fumigatus produces a number of biologically active molecules, including gliotoxin. Those molecules slow ciliary beating and can damage the respiratory epithelium and so possibly influence colonization of the airway by this mold. Nine clinical isolates of A. fumigatus were obtained from the sputum of patients with pulmonary aspergillosis (four cases of chronic necrotizing pulmonary as- pergillosis and one case of aspergilloma) and applied to cultured respiratory ciliated epithelium obtained from the nasal mucosa of healthy volunteers. CBF was measured with a photometric technique that used a phase-contrast microscope. Eight of the nine filtrates from cultures of A. fumigatus isolates caused a significant decrease in CBF; five of the nine caused at least a 50% decrease. A gliotoxin metabolite coeluted with gliotoxin. An extract similar to but with a slightly different absorbency from laboratory-grade gliotoxin also coeluted; this indicates the presence of other, as yet unidentified ultravio- let-absorbing material in the ciliotoxic fraction. Gliotoxin has been shown to inhibit phagocytosis by rodent macrophages, bactericidal activity of perito- neal macrophages, and the basal rate of hydrogen peroxide production by human neutrophils (Eichner et al., 1986). Immunotoxicity Immunotoxicity can result from immunosuppression after exposure to a xenobiotic or from immunoenhancement, autoimmunity, and allergic reactions. Immunosuppression can increase susceptibility to infectious dis- ease 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 alveo- lar macrophages in in vitro studies. Some of those studies are summarized here. Alveolar macrophages (called pulmonary macrophages in some stud- ies) 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

TOXIC EFFECTS OF FUNGI AND BACTERIA 151 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 ani- mals generally examine the effects of short-term exposures. Table 4-3 lists some immunoactive mycotoxins, the fungi that produce them, their po- tency expressed by various measures, their mechanisms of action (if known), and their 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 immunosuppres- sive 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) pre- vents 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 mea-

152 TABLE 4-3 Immunoactive Mycotoxins and Effects Mycotoxin Producing Molds Potency Mechanism Effect Aflatoxin Aspergillusfiavus, 0.6–10.0 ppm in feed: Inhibits protein synthesis Immune Aspergillus depressed antibody suppression— parasiticus response in chickens various end points Ochratoxin A Aspergillus 5 mg/kg ip in mice: Inhibits φ-alanyl tRNA Suppression (OTA) ochraceus, subchronic T-cell synthetase of antibody Penicillium suppression production and verrucosum, globulin synthesis Penicillium viridicatum Sterigmatocystin Aspergillus Inhibits φ-alanyl tRNA Suppression versicolor, synthetase and lipid of antibody Aspergillus peroxidation production and nidulans globulin synthesis, proliferation of Kupffer cells in liver Gliotoxin Aspergillus Inhibits MP phagocytosis, Infection with A. fumigatus induces MP apoptosis, fumigatus, other blocks T- and B-cell microorganisms activation Cyclopiazoic acid Aspergillus spp., Causes lymphoid Immunosuppression (CPA) Penicillium spp. depletion, inhibits protein synthesis

Citrinin Penicillium spp. 0.12–3.9 mg/kg ip mice: Stimulates mitogen Immune modulating lymphocyte production blastogenesis Patulin Aspergillus spp., 10 mg/kg in rats: Inhibits protein synthesis, Immune modulating Penicillium spp. decrease IgA impairs MP activity Trichothecenes Stachybotrys T-2 toxin: 0.1 µg/kg Inhibits protein synthesis, Immunosuppression, chartarum; oral in monkeys: severe inhibits peptidyl immune modulating, Trichoderma immunosuppression synthesis, causes decrease in host viride lymphoid necrosis, causes resistance to infection Stachybotrytoxin: LD50 dysregulation of IgA 0.1 mg/kg ip in mice production Cyclosporin A Stachybotrys IC50 = 26.8 ng/mL Inhibits lymphocyte Immunosuppression (CsA) chartarum, proliferation, IL-2 and Streptomyces interferon production, sutubaensis protein folding Zearalenone Fusarium spp. 10 ppm oral in mice: Inhibits DNA synthesis, Immune modulating decreased resistance to lymphoblastogenesis listerosis Rapamycin Streptomyces Inhibits signal Immune modulating hygroscopicus transduction, preventing T-cell activation NOTE: Abbreviations: IC50, inhibitory concentration50, concentration at which 50% of population shows immune change; ip, intraperitoneal injection; LD50, lethal dose50, dose that kills 50% of population; MP, macrophage. 153

154 DAMP INDOOR SPACES AND HEALTH sured 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 measur- ing 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 in- flammatory alterations occurred as a result of inhalation or intratracheal instillation of AFB1. Those experiments indicate that suppression of alveo- lar 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 phago- cytosis 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 chal- lenged 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 admin- istration of trichothecenes in animals induces more susceptibility to micro- bial 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 isola- tor for 2 years and compared them with 20 nonexposed controls. Histo- logic 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 ani- mals 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 carci- noma 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.

TOXIC EFFECTS OF FUNGI AND BACTERIA 155 In another study, Sumi et al. (1994) exposed germ-free rats to A. versi- color 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 mac- rophages expressed IL-1, IgA antigens, and intercellular adhesion molecules intensely bound to lymphocytes. Numerous lymphocytes infiltrated granu- lomatous 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 micro- bial infection. Acute and chronic exposures to trichothecene mycotoxins result in deple- tion 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 pro- teins and immunoglobulin profiles, reduced antibody formation, thymic apla- sia, reduced cell-mediated immune responses, increased delayed cutaneous hypersensitivity, and impaired bacterial clearance and acquired immunity (Pier and McLoughlin, 1985). Trichothecene immunosuppression also ap- pears 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 re- ductions 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

156 DAMP INDOOR SPACES AND HEALTH has been shown experimentally to produce thymic aplasia, to reduce T-cell function and number, to diminish antibody response, to suppress phago- cytic 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 cul- tured 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 phago- cytic activity of human monocytes was dose-dependent and was signifi- cantly 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 sig- nificantly 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 expo- sure to AFB1 (1–50 µg/mL); greater inhibition was seen after a 48-h expo- sure. The inhibition was time- and dose-dependent. Chromosomal aberra- tions—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. 2 The coating of a particle with a substance that helps it to attach to a phagocytic leukocyte.

TOXIC EFFECTS OF FUNGI AND BACTERIA 157 • 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 cu- taneous 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 concentra- tions 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 macro- phages, 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 cen- tral nervous system symptoms—such as fatigue, headache, memory loss, depression, and mood swings—that they attribute to the indoor environ- ment. 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 neuro- logic 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.

158 DAMP INDOOR SPACES AND HEALTH TABLE 4-4 Neurotoxic Mycotoxins and Effects Mycotoxin Producing Molds Potency Penitrem A Penicillium cyclopium, 250 µg/kg ip in mice: Penicillium tremors, LD 50 = 1.05 verruculosum, mg/kg; 24–25 µg/kg Penicillium crustosum orally in sheep: lethal; 2.2–4.4 µg/kg iv: tremors Penitrem E Penicillium crustosum 2.25 mg/kg ip in mice: tremors Aflatrem Aspergillus flavus 0.5 mg/kg ip in mice: tremors Roquefortine Penicillium commune, 0.1 potency of Penicillium palitans, penitrem A Penicillium crustosum Verruculogen Penicillium 3.0–4.0 µg/kg oral in verruculosum Peyronel sheep: tremors; 13.3 µg/kg oral: lethal; LD50 = 2.4 mg/kg ip Penicillium ED50 = 0.39 simplicissimum mg/kg ip in mice Penicillium crustosum Aspergillus caespitosus Verrucosidin Penicillium 4 mg/kg ip in mice verruculosum var. cyclopium Fumitrem B Aspergillus fumigatus 0.1 potency of Verrucologen Cyclopiazonic Penicillium cyclopium, 250 µg/kg ip in mice: acid Aspergillus flavus, tremors; 2.5 mg/kg: Penicillium crustosum clonic convulsions, death

TOXIC EFFECTS OF FUNGI AND BACTERIA 159 Mechanism Effect References Inhibits Tremors, convulsions Peterson and Penny, 1982; neurotransmitter Selala et al., 1989; Smith, release (GABA) in 1997; Wilson et al.,1968 interneurons of anterior horn of spinal cord Inhibits Tremors, convulsions Kyriakidis et al., 1981 neurotransmitter release (GABA) in interneurons of anterior horn of spinal cord Inhibits Hyper reactivity to Selala et al., 1989; neurotransmitter auditory and ºtactile Wilson et al., 1968 release (GABA) in stimuli, severe interneurons of whole-body tremors anterior horn of spinal cord Motor dysfunction in Norris et al., 1980; Peterson mice, convulsions and Penny, 1982; Selala et al., 1989 Tremors Fayos et al., 1974; Peterson et al., 1982 Paralysis Hodge et al., 1988 Peterson and Penny, 1982 Selala et al., 1989; Wilson et al., 1968 (continued on next page)

160 DAMP INDOOR SPACES AND HEALTH TABLE 4-4 continued Mycotoxin Producing Molds Potency Territrems Aspergillus terreus Citreoviridin Penicillium LD50 = 7.2 citreoviride Biourge, mg/kg ip in Aspergillus terreus, mice Penicillium citreonigrum Ochratoxin A Aspergillus (OTA) ochraceous, Penicillium verrucosum, Penicillium viridicatum Gliotoxin Aspergillus fumigatus Trichothecenes DON Fusarium spp., TDI in infants (for example, other tricothecenes 1.5 µg/kg, in DON, also adults 3.0 µg/kg known as vomitoxin) Stachybotrys chartarum, Trichoderma viride NOTE: Abbreviations: DON, deoxynivalenol; ED50, effective dose50, amount required to produce specified effect in 50% of population; GABA, gamma-aminobutyric acid; IC50, in- hibitory concentration50, concentration at which 50% of population shows immune change; Neurotoxic mycotoxins tend to fall into three general classes: tremor- genic toxins, paralytic toxins, and toxins that interfere with neurotransmit- ters 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 interfer- ing with protein, RNA, or DNA synthesis or that exert their effects on membranes have been examined only for short-term exposures. Susceptibil- ity 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.

TOXIC EFFECTS OF FUNGI AND BACTERIA 161 Mechanism Effect References Tremors, convulsions Inhibits CNS paralysis, convulsions, Franck and Gehrken, 1980; mitochondrial respiratory arrest, death, Selala et al., 1989; ATPases, ATP arrest of respiratory Sorenson, 1993; Steyn synthesis, center (acute cardiac and Vleggaar, 1985; hydrolysis beriberi in humans) Ueno and Ueno, 1972 Competes with Microcephaly (mouse pups), Bruinick and Sidler, 1997; Phe, inhibits sensitive period day 10 of Miki et al., 1994 protein synthesis, gestation affects neural cell differentiation, neuritic Lesions of Chang et al., 1993; astrocytes Jarvis et al., 1995 Inhibits protein Central nervous system or Rotter et al., 1996 synthesis, gastrointestinal effects, increases probably mediated through serotonin vagal receptors because serotonin affects feeding behavior and emesis ip, intraperitoneal injection; iv, intravenous injection; LD50, lethal dose50, dose that kills 50% of population; Phe, phenylalanine; TDI, tolerable daily intake, amount that can be ingested daily without posing significant health risks. 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 in- volves sustained tremors, limb weakness, ataxia, and convulsions (Steyn and Vleggaar, 1985). Tremorgenic toxins generally initiate measurable ef- fects in experimental animals within minutes of exposure. Norris et al. (1980) found that penitrem A (produced primarily by P. crustosum) in- creased spontaneous release of the endogenous neurotransmitters glutamate, γ-aminobutyric acid (GABA), and aspartate by interfering with the neu- rotransmitter-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.,

162 DAMP INDOOR SPACES AND HEALTH 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 cumula- tive 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 pre- natal 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 sensi- tive to OTA than the meningeal fibroblast cultures previously studied by Bruinink et al. (1997); this supports previous suggestions based on in vivo

TOXIC EFFECTS OF FUNGI AND BACTERIA 163 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 phe- nylalanine (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 phenyl- alanyl-tRNA synthetase, and research on rat hepatocytes indicated Phe hydroxylation (Creppy, 1995; Creppy et al., 1983); but these mecha- nisms do not seem to be involved in brain and retinal cell cultures (Bruinink and Sidler, 1997). The differences, however, might be a func- tion 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 af- fects 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. Neuro- toxicity 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 com- monly 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-

164 DAMP INDOOR SPACES AND HEALTH 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 inhibi- tors 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 concen- trations in the cerebellar and brainstem regions; no systemic signs of toxico- sis 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 depres- sion 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 trans- mitter concentrations in the vomiting center in the medulla (Prelusky, 1993). Sensory Irritation The neurotoxic end points that appear to be most affected at low exposures are those which affect the olfactory sense and the “common chemical sense” that responds to pungency (Cometto-Muniz and Cain, 1993; Korpi et al., 1999; Pasanen et al., 1999; Schiffman et al., 2000). It is thought that the common chemical sense resides in the trigeminal, vagus, and glossopharyngeal spinal nerves. Experiments suggest that the sensory

TOXIC EFFECTS OF FUNGI AND BACTERIA 165 nerve endings respond to irritative stimuli, whereas the motor portion re- sponds by smooth muscle contraction, secretion from excretory glands, and central nervous system effects that can include impairment of attention and memory and various fight or flight responses. Perceived pungency can pro- duce reflex constriction of the airways and inflammation and result in nasal stuffiness, headache, malaise, memory loss, and reduced ability to concen- trate, depending on the nature of the irritant, its concentration, and indi- vidual sensitivity (Cometto-Muniz and Cain, 1993; Kasanen et al., 1998; Lucero and Squires, 1998). In animals, the 50% respiratory dose (RD50, the concentration that causes a 50% decrease in respiratory rate in exposed animals, in this case, in response to trigeminal nerve stimulation by a pun- gent chemical) varies for different enantiomers of pinene (a terpene pro- duced by some microorganisms); this indicates that sensory irritant recep- tors respond to the three-dimensional structures of such pungent nonreactive molecules (Korpi et al., 1999). Microorganisms can produce volatile organic compounds (VOCs). Some microbial VOCs or MVOCs (such as alcohols, aldehydes, and ke- tones) are products of primary metabolism and are produced throughout an organism’s life. Others, which tend to be more complex, have character- istic moldy, musty, or pungent odors. They are produced through second- ary metabolism—in Penicillium and Aspergillus—around the time of sporu- lation, when mycotoxins also tend to be produced (Fiedler et al., 2001; Larson and Frisvad, 1994). VOCs produced by building materials, paints, solvents, and combustion can irritate the mucous membranes of the eyes and respiratory tract and possibly the nerve endings of the common chemi- cal sense either alone or in concert with other volatile and semivolatile compounds produced by microorganisms (Otto et al., 1990; Schiffman et al., 2000). Miller et al. (1988) measured a total putative MVOC concentra- tion of 2 mg/m3 in a “moldy” building. Controlled human experiments indicate that aggregate exposure to non-microbial MVOCs common to new office buildings at a total concen- tration of 25 mg/m3 produced subtle changes in some measurable neuro- psychologic end points (Hudnell et al., 1992; Otto et al., 1990, 1992). A companion study by Koren et al. (1992) also found increased neutrophils, a sign of inflammation, in 14 volunteers exposed to the VOC mixture, an indication that such VOCs can elicit an inflammatory response. MVOCs include terpenes, sesquiterpenes, and other substances that are highly irri- tating, but it is unknown whether the concentrations of MVOCs and semivolatile compounds typically found in homes with microbial contami- nation are sufficient to cause a trigeminal or toxic response (Ammann, 1999; Korpi et al., 1999).

166 DAMP INDOOR SPACES AND HEALTH Dermal Toxicity Indoor surface contamination with molds is common and, because dermal absorption can occur, it is possible that surfaces with large amounts of contamination might provide a means of exposure to occupants or work- ers who come into contact with such surface contamination. Simple and macrocyclic trichothecenes are irritating to the skin of ani- mals and humans. Buck and Cote (1991) describe the effects as radiomi- metic in potency. A dose as low as 0.5 ng can cause skin reddening in guinea pigs (Ueno, 1984a). In general, type A (such as T-2 toxin) and type D (such as verrucarin A and satratoxins) trichothecenes are highly irritat- ing, and type B (such as deoxynevalinol) trichothecenes less so. All those cause skin reddening in early stages of toxicity, but type D trichothecenes are characterized by edematous damage to skin tissue (Ueno, 1984b). Large volumes of inflammatory exudate containing lower concentrations of so- dium and proteins and greater amounts of potassium, calcium, and phos- phorus than serum, accumulate in skin tissue; macrocyclic trichothecenes apparently increase the permeability or leakiness of blood vessels (Ueno, 1984a). Trichothecenes from Stachybotrys atra (S. chartarum) were isolated from contaminated insulation and ductwork in a house. Workmen han- dling the material without skin protection suffered painful skin lesions on their hands, armpits, and genitals (Croft et al., 1986; Jarvis, 1990). Hayes and Schiefer (1979) characterized the effect of small doses of T-2 toxin and DAS in the skin of rats and rabbits as an acute inflammatory reaction that involved hyperemia, edema, and neutrophil exudation, with variable amounts of necrosis of the epidermis. Pang et al. (1987) applied T-2 toxin at 0 and 15 mg/kg in DMSO topically to the skin of SPF juvenile male pigs that had been immunized subcutaneously with sheep red blood cells. Serum samples and whole blood taken periodically were evaluated for clinical pathologic and immunologic changes. Treated pigs displayed anorexia, lethargy, posterior weakness and paresis, persistent high fevers, and reduced weight gain. Neutrophilia, de- creased serum glucose, decreased albumin, decreased alkaline phosphatase activity, and increased serum globulin were seen in treated pigs. In addition to severe local dermal injury, this (sublethal) dose of T-2 toxin caused significant systemic effects, including cellular immune responses. Carcinogenesis Some bacteria and molds found in indoor environments produce mol- ecules 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-

TABLE 4-5 Carcinogenic Effects of Mycotoxins Producing Carcinogenic in Potency or Mycotoxin Molds Mechanism Humans Regulated Levels Aflatoxin Aspergillus AFB 1 activated to Liver, lung cancer Canada: VSD (1 × (AFB1 ) Flavus, epoxide by cytochrome cocarcinogen with 10 –5) = 0.14 ng/kg- Aspergillus P-450 enzyme, DNA- hepatitis B, Epstein-Barr day; Swiss parasiticus, adduct formation viruses with other toxins standards in milk, Penicillium cheese puberulum Sterigmatocystin Aspergillus Similar to AFB1, but Liver tumors, rats, mice; LD50 = 60–800 versicolor, much less potent lung cancer, rats (chronic: mg/kga due to low Aspergillus 2 years, 6+/–4 solubility in water flavus, conidiospores in air) or gastric juices) Penicillium luteum, Bipolaris spp. Ochratoxin A Aspergillus Single-strand DNA Suspect in kidney, pelvis, Canada: VSD (1 × (OTA) ochraceus, breaks, unscheduled urethra, bladder carcinomas 10 –5) = 0.18 ng/kg- Penicillium DNA synthesis, DNA- in Balkans; IARC possible day verrucosum, adduct formation, human carcinogen Aspergillus damages chromosomes alutaceus, Penicillium viridicatum, Penicillium cyclopium (continued on next page) 167

TABLE 4-5 continued 168 Producing Carcinogenic in Potency or Mycotoxin Molds Mechanism Humans Regulated Levels Zearalenone Fusarium Damages chromosomes, Suspected carcinogen (also Canada VSD (ZEN) graminearum DNA adducts in kidney estrogenic properties) (1 × 10 –6) = 0.05 and liver µg/kg-day Citrinin Penicillium Nephrotoxic, mildly NA LD50 = 50 mg/kg in citrinum, hepatotoxic rats; 35–58 mg/kg Penicillium ip and 110 mg/kg verrucosum, orally in miceb 19 Penicillium mg/kg ip in rabbits vindicatum, Aspergillus terreus Patulin Penicillium NA US FDA: 50 µg/kg expansum, of body weight per Penicillium day patulum, Aspergillus terreus Penicillin Acid Aspergillus Affects heartc Hepatocarcinogen in some UK: 20–50 µg/kg of ochraceus animalsc food Luteoskyrin Penicillium Hepatotoxic, Carcinogend islandicum Nephrotoxicd NOTE: Abbreviations: LD50, lethal dose50, the dose that kills 50% of the population; VSD, virtually safe dose. aTerao, 1983. bScott, 1977. cReiss, 1988. dUraguchi et al., 1972.

TOXIC EFFECTS OF FUNGI AND BACTERIA 169 tagenic or clastogenic in various species. Others are transformed to carcino- genic 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 cli- mates. Contamination with AFB1 is associated with high rates of hepa- tocarcinoma in some African countries and appears to potentiate the he- patocarcinogenic properties of hepatitis B virus through its immunotoxic effects (Autrup et al., 1987; Badria et al., 1999; Bechtel, 1989; Groopman et al., 1992). 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. viridi- catum, verrucosum, and cyclopium, which are fairly common contami- nants of grain and other foodstuffs. Although molds producing ochratoxins are occasionally found growing indoors, no study of their potential carcino- genic 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

170 DAMP INDOOR SPACES AND HEALTH 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 expo- sures to grain or peanut dust that contained spores with AFB1 at concentra- tions 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 informa- tion presented in this chapter, the committee has reached several findings and recommendations and has identified several research needs regard- ing the nonallergic effects of molds and bacteria found in damp indoor environments. • Molds that can produce mycotoxins under the appropriate environ- mental 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 micro- bial 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 po- tency 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 expo- sures 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

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Almost all homes, apartments, and commercial buildings will experience leaks, flooding, or other forms of excessive indoor dampness at some point. Not only is excessive dampness a health problem by itself, it also contributes to several other potentially problematic types of situations. Molds and other microbial agents favor damp indoor environments, and excess moisture may initiate the release of chemical emissions from damaged building materials and furnishings. This new book from the Institute of Medicine examines the health impact of exposures resulting from damp indoor environments and offers recommendations for public health interventions.

Damp Indoor Spaces and Health covers a broad range of topics. The book not only examines the relationship between damp or moldy indoor environments and adverse health outcomes but also discusses how and where buildings get wet, how dampness influences microbial growth and chemical emissions, ways to prevent and remediate dampness, and elements of a public health response to the issues. A comprehensive literature review finds sufficient evidence of an association between damp indoor environments and some upper respiratory tract symptoms, coughing, wheezing, and asthma symptoms in sensitized persons. This important book will be of interest to a wide-ranging audience of science, health, engineering, and building professionals, government officials, and members of the public.

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