Review article

The biocontaminants and complexity

of damp indoor spaces: more than

what meets the eyes

Jack D Thrasher and Sandra Crawley

Abstract

Nine types of biocontaminants in damp indoor environments from microbial growth are discussed: (1)

indicator molds; (2) Gram negative and positive bacteria; (3) microbial particulates; (4) mycotoxins; (5)

volatile organic compounds, both microbial (MVOCs) and non-microbial (VOCs); (6) proteins; (7) galactomannans;

(8) 1-3-bD-glucans (glucans) and (9) lipopolysaccharides (LPS – endotoxins). When mold species exceed

those outdoors contamination is deduced. Gram negative bacterial endotoxins, LPS in indoor environments,

synergize with mycotoxins. The gram positive Bacillus species, Actinomycetes (Streptomyces, Nocardia and Mycobacterium),

produce exotoxins. The Actinomycetes are associated with hypersensitivity pneumonitis, lung and

invasive infections. Mycobacterial mycobacterium infections not from M. tuberculosis are increasing in immunocompetent

individuals. In animal models, LPS enhance the toxicity of roridin A, satratoxins G and aflatoxin B1

to damage the olfactory epithelium, tract and bulbs (roridin A, satratoxin G) and liver (aflatoxin B1). Aflatoxin

B1 and probably trichothecenes are transported along the olfactory tract to the temporal lobe. Co-cultured

Streptomyces californicus and Stachybotrys chartarum produce a cytotoxin similar to doxorubicin and actinomycin

D (chemotherapeutic agents). Trichothecenes, aflatoxins, gliotoxin and other mycotoxins are found in

dust, bulk samples, air and ventilation systems of infested buildings. Macrocyclic trichothecenes are present

in airborne particles <2 mm. Trichothecenes and stachylysin are present in the sera of individuals exposed

to S. chartarum in contaminated indoor environments. Haemolysins are produced by S. chartarum, Memnoniella

echinata and several species of Aspergillus and Penicillium. Galactomannans, glucans and LPS are upper and lower

respiratory tract irritants. Gliotoxin, an immunosuppressive mycotoxin, was identified in the lung secretions

and sera of cancer patients with aspergillosis produced by A. fumigatus, A. terreus, A. niger and A. flavus.

Keywords

Bacteria, construction defects, mold, mycotoxins, particles

Introduction

Damp or wet building materials occur from a variety

circumstances: water intrusion from floods, hurricanes,

construction defects, roof leaks, condensation,

appliance and plumbing leaks, poorly designed

foundations, etc. Furthermore, building materials can

become wet during storage, transportation and/or

construction. For simplicity, we will use the phrase

water intrusion’ as an all encompassing term.

Water intrusion into buildings permits amplification

of growth of fungi, bacteria and protozoa

(Andersson et al., 1997; Gorny, 2004; Gorny et al.,

2001, 2002; Hirvonen et al., 2005; Peltola et al.,

2001a,b; Rintala et al., 2001, 2002, 2004, Yli-Pirila

et al, 2004). The increased health risks and economic

impact from microbial growth resulting from indoor

dampness are recognized as significant public health

problems requiring attention and remediation (Bernstein

et al., 2008; Cox-Ganser et al., 2005; Fisk

et al., 2007; Genuis, 2007; Mudarri and Fisk, 2007;

Nevalainen and Seuri, 2005). The bio-contamination

resulting from water intrusion includes: (1) molds;

(2) bacteria; (3) microbial particulates; (4) mycotoxins;

(5) volatile organic compounds (non-microbial

Corresponding author:

Jack D. Thrasher, 6635 Sylvan Road, 1011 Citrus Heights, CA

95610, USA. Email: toxicologist1@msn.com

Toxicology and Industrial Health

25(9-10) 583–615

ª The Author(s) 2009

Reprints and permission: http://www.

sagepub.co.uk/journalsPermission.nav

DOI: 10.1177/0748233709348386

tih.sagepub.com

583

[MVOCs] and microbial [VOCs]); (6) proteins (e.g.

secreted enzymes, haemolysins and siderophores);

(7) galactomannans (extracellular polysaccharides or

EPS); (8) 1-3-Db-glucans (glucans) and (9) endotoxins

(lipopolysaccharides [LPS]). In this communication,

we review indoor biocontaminants resulting

from water intrusion and their associated toxicity to

animals and humans. It is apparent that the potential

additive and synergistic effects of multiple contaminants

in the indoor environment have been largely

overlooked, except in experimental animal models

(Huttunen et al., 2004; Isalm and Pestka, 2006; Islam

et al., 2002, 2007; Zhou et al., 1998, 1999, 2000). The

study of health risks to humans from exposure to molds

have been limited to respiratory disease (asthma) in

adults and children (Antova et al., 2008; Jaakkola and

Jaakkola, 2004; Rydjord et al., 2008). In this paper, we

also review the peer-reviewed research that points to

the impacts on human health, including neurological,

respiratory and immune systems and other organs,

from exposure to damp indoor spaces.

Molds

Fungal contamination as a major contributor to sick

building syndrome has been reviewed. The significant

factors for mold growth are water, temperature and

substrate (Li and Yang, 2004). Water activity (aw)

represents available water in a substrate. It is

expressed as a decimal fraction of the amount of water

present in a substrate that is in equilibrium with

relative humidity. Molds that grow at various aw are

classified as xerophilic (xerotolerant), mesophilic and

hydrophilic. The xerophilic molds include species of

Penicillium, Aspergillus and Eurotium that grow at

aw <0.8. Mesophilic molds grow at aw 0.8-0.9 and

include Alternaria, Cladosporium, Phoma, Ulocladium

and Epicoccum nigrum. The hydrophilic molds

include Chaetomium globosum, Fusarium, Stachybotrys

chartarum, Memnoniella echinata, Rhizopus

stolonifer and Trichoderma spp. at aw > 0.9. Thus, the

genera of mold identified indoors are indicative of

the extent of water intrusion. Ergosterol and mycotoxins

are indicators of mold growth (Hippelein and

Rugamer, 2004; Li and Yang, 2004).

Molds grow on surfaces as well as in hidden areas

such as in carpet, behind wall paper, inside interior

and exterior walls, in attics, in subflooring, etc. They

thrive on wet building materials rich in carbohydrates.

Molds take nutrients from dead organic material

(wood, dry wall, paint, paper, glues, etc.) by secreting

digestive enzymes into the matrix upon which they

are growing. It is estimated that approximately 50%

of the mold growth in damp indoor environments is

hidden, e.g. within walls cavities, carpets, etc. Molds

are present in the ventilation systems of contaminated

homes, buildings and automobiles (Ahearn et al.,

1996, 2004; Li and Yang, 2004).

Certain species of molds are more abundant

(amplified) indoors vs outdoors. These include Aspergillus

flavus, versicolor, sydowii, niger and fumigatus

and Penicillium chrysogenum, brevicompactum,

citrinum and decumbens, Chaetomium, Epicoccum,

Fusarium and S. chartarum. Cladosporium species

are often equally abundant outdoors and indoors. The

comparison of total mold spore counts from indoor to

outdoor samples is not an adequate method to test for

mold contamination. Air sampling is only a snapshot

of an indoor environment that fluctuates according to

various parameters, e.g. human activity, air conditioning,

temperature, opened/closed windows, etc. The

extent of mold and other microbial growth must be

determined from a combination of samples that

include bulk, wipe, air, carpet dust and wall cavity

sampling. Next, the frequency (percentage) of various

species of Aspergillus, Penicillium, Stachybotrys,

etc., in the indoor vs the outdoor samples must be

determined. This approach will reveal that several

of the aforementioned molds have amplified indoors

when compared to outdoors. The profile of indoor

mold may be constant when compared to the outdoor

profile. However, certain species of Aspergillus and

Penicillium, as mentioned above, will be dominant

indoors vs outdoors (Schwab and Straus, 2004; Straus

et al., 2003; Wilson and Straus, 2002). For example,

the authors have observed situations in which Penicillium

species were at 100% in the indoor air and bulk

samples, while outdoor levels were <12%. Moreover,

in other samples, Aspergillus species were greater

indoors (46%) vs outdoors (<6%). The US EPA has

developed the polymerized chain reaction (PCR)

DNA technology to identify 130 of the major indoor

fungi to the species level and has licensed several

companies to utilize the method (USEPA, 2007).

As an example, the role of air speed equivalent to

normal human activity on the release of spores from

mold colonies has been reported in bench studies

(Gorny 2004; Gorny et al., 2001, 2002; Tucker

et al., 2007). Low air speeds cause an initial release

of spores from colonies of S. chartarum, Aspergillus

niger and versicolor; Penicillium chrysogenum and

melinii, and Cladosporium sphaerospermum and

584 Toxicology and Industrial Health 25(9-10)

584

cladosporioides. The spore release per square

centimeter during the first 10 min was lowest for

C. cladosporioides (<500 spores); followed by

S. chartarum (4000 spores) and Aspergillus and Penicillium

spp. (10,000 spores each). After the initial

release, additional spore releases did not occur with

Stachybotrys and Cladosporium, while those of

Aspergillus and Penicillium slowly declined for the

duration of the 70 min of observations (Tucker

et al., 2007). The proportion of released amounted

to 0.2% (S. chartarum); 0.8% (C. cladosporioides);

1.1% (A. niger) and 1.8% (P. chrysogenum) of total

spore mass of each mold. These observations demonstrate

that dry spore molds (Aspergillus and Penicillium)

more readily release their conidia when

compared to sticky clusters of spores of S. chartarum.

Moderately to heavily damaged homes in the aftermath

of Katrina had elevated levels of Aspergillus,

Penicillium and Paecilomyces (Chew et al., 2006;

Rao et al., 2007b). Moreover, molds detected in

water-damaged building materials include A. versicolor,

A. sydowii, Trichoderma viride, S. chartarum,

Chaetomiumglobosum,multiple species of Penicillium,

Acremonium, Cladosporium, Phoma, Aureobasidium,

Phialaphora and yeast (Reijula, 2004).

Rodent lungs have been used to test the adverse

effects of various components of common indoor

molds. Proteases, isosatratoxin-F, and spores of S.

chartarum cause inflammatory and cytotoxic effects

in the lungs of juvenile mice. Among the adverse

effects are alterations and morphological changes in

Type II alveolar cells (Rand et al., 2002), release of

interleukin 1b (IL-1b), IL-6, IL-8 and tumor necrosis

factor a(TNF-a) with neutrophilia, granuloma and

reduced collagen IV (Pestka et al., 2008; Yike et al.,

2007) and a decrease in alveolar space (Rand et al.,

2003). Moreover, spores from indoor species of

Aspergillus and Penicillium cause lung eosinophilia,

neutrophilia, release of inflammatory cytokines

(IL-6, TNF-a), vascular leakage, elevated LDH,

Th-2 inflammatory responses and other cytotoxic

damage in mouse lungs (Cooley et al., 1998, 2000,

2004; Jussila et al., 2002b; Schwab and Straus, 2004).

S. chartarum consists of two chemotypes: one

produces trichothecene mycotoxins, while the other

releases spirocyclic atranones. Both cause inflammation

in mouse lungs (Flemming et al., 2004). S. chartarum is a

slimy greenish black mold that does not readily release

spores. Thus, the presence of its spores in the indoor air

and bulk samples signals either dried disturbed colonies

and/or contamination. The spores of Stachybotrys are

rare findings in outdoor air (Cooley et al., 1998; Li and

Yang, 2004; Schwab and Straus, 2004; Shelton et al.,

2002).

Another issue that has been overlooked is the role

of molds in chronic upper and lower respiratory tract

disease. Chronic rhinosinusitis (CRS) appears to be a

non-immunoglobulin E (IgE) immunological inflammatory

response to fungi with nasal eosinophilia and

the release of toxic major basic protein has a favorable

response to intranasal amphotericin B (Kern et al.,

2007; Ponikau et al., 2005, 2006; Sasama et al.,

2005). Cladosporium, Aspergillus, Alternaria and

Penicillium were frequently cultured from nasal

polyps with a histologic type of fibro inflammation

present in over 60% of patients vs controls. Fungi

were commonly cultured during the hot and humid

environment of summer time from the polyps (Shin

et al., 2007). In addition, Alternaria, Aspergillus and

Cladosporium proteases interact with nasal epithelial

cells activating protease receptors (PARs 2 and PARs

3) enhancing the production of chemical mediators

and migration of eosinophils and neutrophils into

nasal polyps (Shin et al., 2006). The condition

involves exaggerated humoral response of both TH1

and TH2 types. Peripheral mononuclear blood cells

(PMBCS) from CRS patients produce significantly

elevated IL-5 and IL-13 to Alternaria and Cladosporium

antigens and increased levels of IgG antibodies to

Alternaria (Shin et al., 2004). Also, the antimicrobial

peptide, cathelicidin LL-37, is up-regulated by antigens

of Aspergillus (fourfold) and Alternaria (sixfold)

in CRS patients as well as up-regulated surfactant

protein (Ooi et al., 2007a,b).

Molds may infect and/or colonize. Rao et al.,

(2007a) reported eight cases of colonization of New

Orleans immunocompetent residents with Syncephalastrum.

The organism was isolated from clinical

specimens of sputum, BAL, endotracheal aspirates

and nasal swabs. Also, the existence of aspergillosis

in immunocompetent and immunocompromised

humans is not questioned (Lewis et al., 2005a,b; Raja

and Singh, 2006; Samarakoon and Soubani, 2008;

Strelling et al., 1966). Several other zygomycetes are

capable of causing human disease (Ribes et al., 2000).

Mucormycosis of immunocompetent individuals

with involvement of the gastrointestinal tract, skin,

paranasal sinuses, necrotizing fasciitis and pericardium

has been described in India (Jain et al., 2006;

Prasad et al., 2008).

Pulmonary aspergillosis and clinical update of the

disease has been recently reviewed (Zmeili and

Jack D Thrasher and Sandra Crawley 585

585

Soubani (2007). The recognized clinical conditions

are aspergilloma, pulmonary aspergillosis (non-invasive),

invasive aspergillosis (IA), chronic necrotizing

aspergillosis (CNA) and allergic bronchopulmonary

aspergillosis (ABPA). Aspergilloma (fungus ball)

occurs in individuals with pre-existing lung disease

(tuberculosis, sarcoidosis, bronchiectasis and cysts).

The fungus ball exists in pre-existing cavities in

diseased lungs. It contains fungal hyphae, inflammatory

cells, fibrin, mucous and tissue debris. Other

organisms causing fungal balls are zygomycetes and

fusarium. Diagnosis of pulmonary aspergilloma is

usually based upon clinical history and radiographic

features. Approximately 50% of sputum cultures are

positive for fungi. The development of IA is usually

rapid in immunocompromised individuals (e.g. cancer

chemotherapy, organ transplant and haematopoietic

stem-cell transplantation [HSCT]) with high mortality

in neutropenic (50%) and HSCT (90%) patients. CNA

is semi-invasive, resulting from infections of the

lungs and runs a slow progressive course. APBA is

hypersensitive to Aspergillus antigens usually in

individuals with asthma or cystic fibrosis who are

dependent upon chronic corticosteroid therapy.

APBA is an inflammatory condition involving

hypersensitivity Type I and III immune responses.

The pathology is poorly understood while chronic

granulomatous conditions exist in the lungs of

affected individuals.

IA by A. fumigatus have been reported in immunocompetent

children. Strelling et al., (1966) describe

the deaths of a brother (14 months) and sister (4 years)

from acute pulmonary aspergillosis and review the

literature. The two children developed acute fever,

cough and breathlessness with yellowish-brown

(haemoptysis) mucus, after playing in a barn. Cultures

from the lungs and barn materials isolated A. fumigatus,

while tissue sections of the lungs revealed branching

fungal hyphae. The doctors’ review of the

literature described additional deaths and disease

from aspergillosis as follows: (1) brother (5 years) and

sister (11 years) living on a farm; (2) 20-day-old

infant; (3) 18-day-old infant; (4) 7-year-old child (sex

not given); (5) boy (6 years) and (6) girl (7 years) who

recovered. The cerebellum and frontal lobes were

involved in cases 5 and 6.

Immunocompetent adultsmay develop non-invasive

or invasive Aspergillus infections. Non-invasive aspergillosis

involves aspergilloma of the lungs visible on

computed tomography (CT) scan with a solitary nodule

or mass. Microscopically granuloma and cavity lumen

hyphae are present (Kang et al., 2002). On the other

hand, IA may infect the lungs as well as other organs.

The lungs can be almost completely consolidated with

granuloma (Hillerdal et al., 1984; Reijula and Tuomi,

2003), bilateral hilar prominence (Parameswaran

et al., 1999), bilateral fibrinous pleural adhesion and

extensive parenchymal destruction (Zuk et al., 1989).

Blood-stainedmucous in bronchi and massive haemoptysis

may also be present (Parameswaran et al., 1999;

Zuk et al., 1989). Invasion of the tracheobronchial tree

can also occur (Mohan et al., 2006). Individuals with

asthma or chronic obstructive pulmonary disease

(COPD) on either short-term or prolonged corticosteroid

therapy (oral, inhalation, intravenous [i.v.]) contract

IA mostly from A. fumigatus followed in order by

flavus, terreus, niger and nidulans (Ali, et al., 2003;

Ganassini and Cazzadori, 1995; Samarakoon and

Soubani, 2008; Smeenk et al., 1997; Trof et al., 2007).

Disseminated IA to the skin and bone (spondylodiscitis)

in a patient with a pulmonary nodule has been described

(Domergue et al., 2008),while it involved the lung, liver

and spleen in another documented patient (Raja and

Singh, 2006). Paranasal sinus involvement, perforation

of the nasal septum and invasion of the palate was

described in one patient (Khatri et al., 2000; Raja and

Singh, 2006; Samarakoon and Soubani, 2008), while

another had paranasal, orbital involvement with intracranial

extradural extension via the maxillary division

of the trigeminal nerve (Subramanian et al., 2007). IA

can result in dissemination to the central nervous system

(CNS;Garcia et al., 2006; Palanisamy et al., 2005).CNS

complications in one person involved headache, nausea,

motor impairment and cognitive decline due to

progressive cerebellar lesions. After treatment with high

doses of itraconazole (1600 mg/day), the patient

recovered with only mild cerebellar motor impairment

(Palanisamy et al., 2005).

Finally, diabetes mellitus, corticoid steroids

therapy, COPD and antibiotics are risk factors for

developing IA. Corticosteroids are immunosuppressive.

Treated alveolar macrophages (AM) can internalize

mold conidia (Philippe et al., 2003). However, the

corticosteroids inhibit reactive oxidant intermediates,

e.g. NADPH oxidase, as well as the production of

cytokines TNF-a and IL-1 (Kamberi et al., 2002;

Philippe et al., 2003; Taramelli et al., 1996). Although

the conidia are internalized, the killing of conidia by

the inhibited AM is significantly reduced, allowing

the development of IA (Brummer et al., 2001; Gangneux

et al., 2008; Philippe et al., 2003). Corticosteroid

use is a major risk factor for the increase in IA in

586 Toxicology and Industrial Health 25(9-10)

586

non-neutropenic cases (Gangneux et al., 2008; Trof

et al., 2007). The use of corticosteroids to treat IA

becomes more perilous when the toxic metabolite,

gliotoxin, which is produced by several genera of

molds, is considered. Gliotoxin is an immunosuppressive

mycotoxin produced by A. fumigatus, terreus and

niger, several Penicillium species, Trichoderma

virens and Candida albicans. It has been detected in

the sera and lungs of both mice and humans with

aspergillosis (Gardiner et al., 2005; Lewis et al.,

2005a,b). Thus, therapy-induced immunosuppression

probably leads to fungal metabolite immune suppression.

For more information on gliotoxin, see the

below category entitled ‘Mycotoxins’.

Th-1 cell-mediated immunity was believed to be

the major defense against fungal infections, while

Th-2 humoral immunity plays a minor role (Blanco

and Garcia, 2008). However, recent information

suggests that Th-17 cells and IL23/IL17 are important

in Aspergillus infections and fungal pathology

(Romagnani, 2008; Romani et al., 2008; Tesmer

et al., 2008; Zelante et al., 2007, 2008). In this scenario,

IL-23/IL-17/TGF-b worsen the infection, while IL-6

has a protective role. The regulatory pathway in the

pathogenic inflammation to molds involves the kynurenines

(Belladonna et al., 2006; Romani and Puccetti,

2008). In addition, the acute phase long pentraxin 3

(PTX3) appears to have a role in the inflammatory process

caused by molds (Gaziano et al., 2004).

In mice, the Th-17 cell develops from naive CD4 T

cells, while in humans its origin maybe from Tregs or

Th-1 cells (Romagnani, 2008). Th-17 was initially

described in autoimmune mouse models of autoimmunity:

encephalomyelitis (EAE) and collagen-induced

arthritis (CIA). Human Th-17 cells promote disruption

of blood–brain barrier tight junctions, promote CNS

inflammation and kill human neurons through recruitment

of CD4þ lymphocytes (Kebir et al., 2007).

Furthermore, the interleukin, IL-17, secreted by

Th-17 lymphocytes is elevated in the sera and diseased

tissues from various chronic inflammatory diseases:

e.g. Chron’s disease, lupus erythematosus and rheumatoid

arthritis. However, in autistic children, plasma

IL-17 is not elevated, while IL-23 is decreased

(Enstrom et al., 2008). It is also likely that Th-17 cells

and IL-17 are involved in severe asthma complicated

by bacterial infections independent of IgE-mediated

allergic disorders (Romagnani, 2008).

The pentraxins are a family of multimeric patternrecognition

proteins. They are divided into short

(C-reactive protein) and long (PTX3) pentraxins

(Mantovani et al., 2008)). Long PTX3 expression is

induced in response to inflammatory signals at sites

of inflammation. It is secreted by endothelial cells,

monocytes/macrophages, dendritic cells (DCs),

smooth muscle cells, fibroblasts and is stored in

neutrophil granules (Imamura et al., 2007; Jaillon

et al., 2007; Savchenko et al., 2008). Long PTX3 is

expressed in individuals with chronic systemic

inflammation (Muller et al., 2001; Savchenko et al.,

2008). PTX3 is an acute-phase protein produced at

sites of infections and is thought to have a protective

role against microbial infections, e.g. bacteria, fungi,

viruses (He et al., 2007). However, over expression of

long PTX3 is associated with more severe in lung

injury and correlates with the state of severity of critical

illness as well as organ failures (He et al., 2007;

Mauri et al., 2008; Muller et al., 2001; Suliman

et al., 2008). Thus, in a mouse model of aspergillosis

depending upon the therapeutic dose, PTX3 either

exacerbated or improved the state of pulmonary

inflammation (Gaziano et al., 2004). Finally, long

PTX3 is expressed in the CNS of mice following

instillation of LPS and during infections by either

C. albicans or Cryptococcus neoformans (Polentarutti

et al., 2000).

Positive gram and gram negative bacteria

Gram positive and gram negative bacteria have been

isolated from damp indoor spaces. They are briefly

reviewed.

Gram Positive Bacteria

Gram positive bacteria have been isolated from waterdamaged

building materials. Actinomycetes (Actinobacteria)

including several species of Streptomyces,

Nocardia and Mycobacterium were cultured from

indoor air and dust as well as from moldy, waterdamaged

materials (Peltola et al., 2001a,b; Rautiala

et al., 2004; Rintala et al., 2001, 2002, 2004; Torvinen

et al., 2006). Several species of both potentially

pathogenic and saprophytic of Mycobacteria were

isolated from workplace air during remediation (Rautiala

et al., 2004). Some of the identified species

belonged to the Mycobacterium avium complex and

are potential human pathogens. It was noted by the

authors that the mycobacteria are slow growing. The

Actinomycetes are potential human pathogens. The

reporting of these infections in the United States is not

required; therefore, it is impossible to determine disease

prevalence.

Jack D Thrasher and Sandra Crawley 587

587

Streptomyces californicus produces spores approximately

1 mm in mean aerodynamic diameter that can

penetrate deep into alveolar spaces of the lungs.

Intrathecal instillation of spores in mice caused

inflammation characterized by increase concentrations

of TNF-a, IL-6, LDH, albumin and haemoglobin

in bronchoalveolar lavage fluid (BAL) and sera

(Jussila et al., 2001). Moreover, following repeated

exposures to the spores in a dose-response study, the

inflammation was systemic, involving recruitment

of neutrophils, macrophages and activated lymphocytes

into the airways and decreased numbers of

spleen cells. The dose response was non-linear. BAL

from the mice also contained increased concentrations

of albumin, total protein, LDH and activated lymphocytes.

It was concluded that S. californicus spores are

capable of causing both lung inflammation and systemic

immunotoxic effects (Jussila et al., 2002a,

2003). It is interesting that Streptomycetes produce

anthracyclines, e.g. daunorubicin and doxorubicin,

drugs widely used in chemotherapy (Arcamone,

1998; Arcamone and Cassinelli, 1998). The anthracyclines

cause apoptosis of activated and non-activated

lymphocytes as well as a decrease of mature T and B

cells in mice. The T and B cell depletion was most

severe in the spleen, moderate in lymph nodes and

least in the thymus (Ferraro et al., 2000). In addition,

various species of this genus are the source for a

variety of antibiotics (Gunsalus, 1986).

The inflammatory and cytotoxic affects in vitro of

indoor air bacteria compared to mold spores have

been reported. The bacteria tested were Bacillus cereus,

Pseudomonas fluorescens and mold tested were

Streptomyces californicus, A. versicolor, P. spinulosum

and S. chartarum. The bacteria caused the production

of IL-6 and TNF-a in mouse macrophages.

Only the spores of S. californicus caused a production

of nitric oxide (NO) and IL-6 in both mouse and

human cells. Of the molds, only S. chartarum caused

the production IL-6 in human cells. The overall

potency to stimulate the production of proinflammatory

mediators decreased in order as follows: Ps.

fluorescens, > S. californicus, > B. cereus, > S. chartarum,

> A. versicolor, > P. spinulosum. There was a

synergistic response of TNF-a and IL-6 after coexposure

with S. californicus with both trichodermin and

7-a-hydroxytrichodermol. These observations indicate

that bacteria in water-damaged buildings should

also be considered as causing inflammatory effects

on occupants (Huttunen et al., 2004; see Table 1).

The synergism and interaction of S. californicus

and S. chartarum on mouse macrophages have been

reported. Spores from these two organisms were

tested for the effects on macrophages as follows:

spores isolated from co-cultivated cultures, mixture

of the spores from separate pure cultures and the

spores of each organism. Spores isolated from the

co-cultures were compared to the mixture of spores

and were more cytotoxic than either the mixture

of spores or the spores from each organism.

Co-cultured spores caused increased apoptosis of the

macrophages by more than fourfold. Cells arrested at

G2/M stage of the cell cycle were increased nearly

twofold. In contrast, the co-cultured spores significantly

decreased the ability of the spores to trigger the

production of NO and IL-6 by the macrophages. Thus,

co-culturing of the two organisms resulted in microbial

interactions that significantly potentiated the

ability of spores to cause apoptosis and cell cycle

arrest (Penttinen et al., 2005). In a follow-up study,

the same authors (Penttinen et al., 2006) compared the

cytotoxicity of the co-cultured spores (S. californicus

and S. chartarum) to that of chemotherapeutics (doxorubicin,

phleomycin, actinomycin D and mitomycin

C) produced by S. californicus. The co-cultured

spores mediated apoptosis, cell cycle arrest at the

Table 1. Toxic metabolites produced by bacteria isolated from water-damaged materials and indoor air

Metabolite Disease Organisms Health concerns

Valinomycin Unknown Streptomyces griseus Mitochondrial poison

Leptomycin B Unknown Streptomyces species Inhibition of inducible nitric oxide synthetase

Toxic peptide Unknown Bacillus amyloliquefaciens Depolarized trans-membrane. Decreased ATP and

NADH cell death

Mitochondrial toxin Unknown Bacillus pumilus Disruption of mitochondrial membrane

Mitochondrial toxin Unknown Nocardiopsis species Disruption of mitochondrial membrane

Cytostatic compounds Unknown Co-culture of S. chartarum

and S. californicus

Cytotoxic compounds that are just as toxic

as doxorubicin and AMD

588 Toxicology and Industrial Health 25(9-10)

588

S-G2/M phase and caused a fourfold collapse of

mitochondrial membrane potential. In addition, a

sixfold increase in capase-3 activation and DNA

fragmentation was observed. The cytotoxicity of the

co-cultured spores was similar to that caused by doxorubicin

and actinomycin D. It was concluded that the

co-culture of the two organisms caused the production

of unknown cytotoxic compound(s) that evoked

immunotoxic effects similar to chemotherapeutic

drugs. In conclusion, these studies demonstrate that

spores from S. californicus are cytotoxic to mouse

macrophages. More importantly, the co-cultivation

of S. californicus and S. chartarum results in a spore

mixture that is more toxic than the spores of each

organism cultured individually. Additional attention

must be paid to the synergism that probably occurs

in the microbial mixture that is present in waterdamaged

buildings. Moreover, the spores of S. californicus

were more toxic to mouse macrophages than

was a mixture of spores from co-cultures with various

molds (A. versicolor, P. spinulosum and S. chartarum).

S. californicus spores alone were more potent

inducers of inflammatory and cytotoxic responses

than any combination of co-cultivated spore mixtures.

In addition, co-culture of S. chartarum and A. versicolor

produced a synergistic increase in cytotoxicity

with no effect on inflammatory responses of the

macrophages (Murtoniemi et al., 2005). Finally,

S. griseus strains isolated from indoor environments

produce a toxin, valinomycin, which causes mitochondrial

swelling, damaged mitochondrial membranes,

and disrupted the mitochondrial membrane

potential of boar sperm (Andersson et al., 1997;

Peltola et al., 2001a). In addition, the Nocardiopsis

strains isolated from indoor water-damaged environments

are toxigenic and produce a mitochondrial

toxin(s) that damages the mitochondria of boar sperm

(Peltola et al., 2001a,b). In conclusion, these studies

demonstrate that spores from S. californicus are cytotoxic

to mouse macrophages. More importantly, the

co-cultivation of S. californicus and S. chartarum

results in a spore mixture that is more toxic than the

spores of either organism cultured individually. Additional

attention must be paid to the synergism that

probably occurs in the microbial mixture that is

present in water-damaged buildings. Finally, Nocardia

isolated from water-damaged building materials

also cause cytotoxicity.

Streptomyces species are associated with farmer’s

lung disease (allergic alveolitis). Infections (Streptomycosis)

occur most frequently in immunocompromised

individuals and people with diabetes mellitus and/or

corticosteroid therapy. However, co-infection with

Aspergillus in cases of chronic granulomatous disease

following exposure to aerosolized mulch has been

reported (Siddiqui et al., 2007). Diagnosis is difficult

because of mimicry of other diseases (Acevedo et al.,

2008; Che et al.,1989; Kagen et al.,1981; Kapadia

et al., 2007; Kofteridis et al., 2007; Madhusudhan

et al., 2007; Quintana et al., 2008; Roussel et al.,

2005). Finally, Streptomyces spp. can causemycetoma,

a condition most endemic around the Tropic of Cancer,

but also occurs worldwide, in the United States, Asia,

and Latin America (Quintana et al., 2008; Welsh

et al., 2007). The organisms are aerobic, produce chalky

aerial mycelia and granules of different sizes, textures

and colors. Streptomyces do not stain with haematoxylin

and eosin, but are gram positive and acid fast stain.

Nocardia are aerobic and infectious (nocardiosis),

producing pulmonary disease, skin infections, lymphocutaneous

lesions and brain abscesses (Bennett

et al., 2007; Mauri et al., 2002; Shook and Rapini,

2007). The genus contains approximately 15 known

species. The species identified in human pulmonary

and systemic infections include asteroides, pseudobrasilenisis,

otitidis-cavriarum, abscessus, farcinica,

nova, transvalensis (Bennett et al., 2007; Georghiou

and Blacklock 1992; Groves, 1997; Kennedy et al.,

2007; Yourke and Rouah, 2003). N. cyriacigeorgica

recently was identified as an emerging pathogen in

the United States and probably worldwide (Schlaberg

et al., 2008). Lymphocutaneous, subcutaneous

mycetoma with sulphur granules and superficial skin

infections also occur (Shook and Rapini, 2007).

N. asteroides was identified with pneumonia and

empyema (thoracis) in a healthy 40-day-old neonate

after presumed inhalation exposure (Tantracheewathorn,

2004). Nocardia are gram positive and stain

partially with acid fast. Serological tests are not available.

Predisposing factors are immunocompromised

individuals, pre-existing lung disease, corticosteroid

therapy and diabetes mellitus (Bennett et al., 2007;

Georghiou and Blacklock, 1992; Mari et al., 2001).

As occurs with Streptomyces, the disease process can

exhibit mimicry. Case reports of immunocompetent

patients include brain abscesses (Chakrabarti et al.,

2008; Dias et al., 2008; Kandasamy et al., 2008),

spinal cord abscess (Samkoff et al., 2008), mimicry

of metastatic brain tumor (Kawakami et al., 2008),

ventriculitis/choroid plexitis (Mongkolrattanothai

et al., 2008), lymphangitis (Dinubile, 2008), lung

abscesses (Mari et al., 2001; Martinez et al., 2008;

Jack D Thrasher and Sandra Crawley 589

589

Tada et al., 2008), endophthalmitis (Ramakrishnan

et al., 2008) and sternal osteomyelitis with mediastinal

abscess (Baraboutis et al., 2008). It is recommended

that 16s recombinant DNA (rDNA) sequencing should

be used to identify infections of novel bacteria (Woo

et al., 2008).

Mycobacteria are common in moisture-damaged

building materials (ceramic, wood and mineral insulation)

and their occurrence increase with the degree of

mold damage (Rautiala et al., 2004; Torvinen et al.,

2006). They are environmental (soil, water, sewage)

opportunistic gram positive bacteria capable of causing

hypersensitivity pneumonitis as well as cervical

lymphadenitis in children. Mycobacteria have been

isolated from water systems, spas, hot tubs and humidifiers

and are resistant to disinfection (Primm et al.,

2004; Torvinen et al., 2007). The Centers for Disease

Control and Prevention (CDC) has implicated

M. avium, terrae and immunogenum in outbreaks of

hypersensitivity pneumonitis (Falkinham, 2003a,b).

M. terrae isolated from the indoor air of a moisturedamaged

building induced a biphasic inflammatory

response after intrathecal instillation into mouse

lungs. There was an initial increase in TNF-a and

IL-6 at 6 hour to 3 days, followed by a second phase

at 7 to 28 days (Jussila et al., 2002a,b).

The genus Mycobacterium consists of approximately

117 species of which 20 are potential human

pathogens. They cause non-tuberculous mycobacteria

(NTM) lung disease (American Thoracic Society,

2007). M. avium-intracellulare organisms are increasingly

significant pathogens in North America, causing

a pulmonary infection named MAC (M. avium complex).

M. kansasii, chelonae and fortuitum are other

important pathogens (Agrawal and Agrawal, 2007;

Fritz and Woeltje, 2007; Fujita et al., 2002; Kuhlmann

and Woeltje, 2007; Iseman et al., 1985). According to

the American Thoracic Society, 2007, ‘The minimum

evaluation for NTM should include the following: (1)

chest radiograph or, in absence of cavitation, chest

high-resolution computed tomography (HRCT) scan;

(2) three or more sputum specimens stained for acidfast

bacilli (AFG) analysis; and (3) exclusion of other

disorders such as tuberculosis. Clinical, radiographic

and microbiologic criteria are equally important and

all must be present to make a diagnosis of NTM lung

disease. The following criteria apply to symptomatic

patients with radiographic opacities, nodular or

cavitary, or HRCT scan that shows multifocal bronchiectasis

with multiple nodules. These criteria fit best

with M. avium complex (MAC), M. kansasii and

M. abscessus. There is not enough known about

NTM of other species to be certain that these diagnostic

criteria are universally applicable to all NTM

respiratory pathogens. A microbiologic diagnosis

includes one of the following: (1) positive cultures

from two separate expectorated samples; (2) positive

culture from at least one bronchial wash; (3) transbronchial

or other lung biopsy with mycobacteria

histopathologic features. Patients suspected of having

NTM lung disease but who do not meet the diagnostic

criteria should be followed until the diagnosis is

firmly established or excluded. NTM is on the rise

worldwide. Mycobacteria have been isolated from

water-damaged building materials from indoor

environments. Finally, individuals treated with corticosteroids

are at an increased risk.

M. ulcerans is a significant human pathogen that

causes Buruli ulcer (BU). Cases of BU have been

reported worldwide with the greatest burden of

disease occurring in West and Central Africa. Its

transmission source is not fully understood, but it may

be waterborne. The disease is characterized by progressive,

severe necrotizing skin lesions that do not

respond to antimicrobial therapy and may require

either surgical excision or amputation as treatment.

M. ulcerans is an intracellular pathogen. It produces

a polyketide-derived macrolide, mycolactone. Mycolactone

is cytotoxic at 2 ng/mL and is the organism’s

virulence factor. Mycobacterium scrofulaceum and

kansasii and other mycobacteria produce a less cytotoxic

(33 to 1000 mg/mL) lipid chemical when tested

on fibroblast in vitro (Daniel et al., 2004; Yip et al.,

2007). The gram positive toxic organisms identified

in indoor environments also include Bacillus spp,

Nocardia spp. and Streptomyces spp. (Peltola et al.,

2001a,b). Mycobacteria have been isolated from

damp indoor environments (Falkinham, 2003a,b;

Jusilla et al., 2001, 2002a).

Examples of additional gram positive bacteria are

species of Atrhrobacter, Bacillus,Cellumonas, Gordona

and Paeniibacillus (Andersson et al., 1997). Bacillus

simplex and Amyloliquefaciens isolated from moisturedamaged

buildings produce surfactin (lipopeptide) and

peptides that adversely affect cell membranes and

mitochondria (Mikkola et al., 2004, 2007). Finally, there

were elevated concentrations of Staphylococci and

Actinomycetes in a water-damaged home in which a

3-month-old infant died from a Reye’s-like syndrome,

with mitochondrial damage resulting in decreased

enzymatic activity of complexes I-IV. Mitochondrial

DNA mutation testing of the infant resulted in negative

590 Toxicology and Industrial Health 25(9-10)

590

findings for known mitochondrial diseases. This home

also contained several species of Aspergillus, Penicillium

and S. chartarum (Gray et al., in preparation).

Gram negative bacteria: Gram negative bacteria

have also been identified in water-damaged buildings.

These bacteria produce endotoxins (LPS) and are

potentially infectious, particularly species of E. coli,

Enterobacter and Pseudomonas (e.g. aeuroginosa

and other spp.). Other gram negative bacteria are species

of Agrobacterium, Caulobacter, Stenophomonas

and Chryseomonas (Andersson et al., 1997). Gram

negative bacteria are ubiquitous in the environment

and have been isolated from contaminated ventilation

systems, humidifiers, carpeting, drywall, etc., in large

numbers in water-damaged buildings. Pet Fecal

matter from dogs and cats and sewage are two sources

of gram negative bacteria. They release endotoxins

that can cause a variety of symptoms as well as

pulmonary inflammation in building occupants (see

endotoxins; Martinez, 2007a,b; Rylander 2004;

Simpson et al., 2006; Yang, 2004). Finally, bioaersols

of Staphylococcus aureus, multi-antibiotic-resistant

and non-resistant strains, have been isolated from

healthy residential homes (Gandara et al., 2006).

Particulates

Colonies of fungi and bacteria shed particulates into

the indoor air ranging from nanoparticles to 9 mm or

larger. For convenience of this discussion, the

particulates will be divided into large and small

particle fractions. The larger particles are >2 mm consisting

of spores and hyphal fragments. They contain

mycotoxins, antigenic material, enzymes, haemolysins

and other potentially toxic metabolites with

adverse effects on the lungs of mice and rats. Hyphal

fragments of S. chartarum and other genera of molds

probably caused pulmonary bleeding in infants (Dearborn

et al., 2002; Etzel et al., 1998; Flappan et al.,

1999; Novotny and Dixit, 2000). S. chartarum was

isolated from the lung of a child with pulmonary

bleeding and haemosiderosis (Elidemer et al., 1999).

Spores, hyphal fragments and extracts from several

genera of molds cause inflammatory changes in the

lungs and nasal passages (Brieland et al., 2001; Rand

et al., 2002, 2003, 2005; Schwab and Straus, 2004;

Yike et al., 2005, 2007).

The shedding of fine particles (<2 mm) from fungal

and bacterial growth results from human activity, e.g.

radio, TV, talking, walking, etc., which creates

vibrations at frequencies from 1 to 20 Hz and air

movement (Gorny, 2004; Gorny et al., 2002). The

vibrations along with air velocity release spores,

hyphal fragments, and fine particulates. The fine

particles are up to 320 times more numerous than the

large particulates, depending upon mold species and

velocity of air. Furthermore, the fine particulates also

contain biocontaminants (mycotoxins, endotoxins,

antigens, haemolysin, etc.) produced by fungi and

bacteria (Brasel, et al., 2004, 2005a,b; Van Emon

et al., 2003) and are immunogenic (Gorny, 2004).

The mean diameter, size and density of particulates,

particularly nanoparticles, determine the deposition in

the nasal cavity and levels of the tracheobronchial tree.

Particles <5 mm are deposited in alveolar spaces

(Oberdorster et al., 2004; Peters et al., 2006). Nanoparticles

mainly deposit in alveoli and transfer into the

systemic circulation and distribute to other organs,

including the brain (Calderon-Garciduenas et al.,

2004, 2008a,b,c; Elder and Oberdorster, 2006;

Nemmar et al., 2001; Peters et al., 2006). Transfer into

the brain via the olfactory nerve is probable withmycotoxins

as well as cadmium, other chemicals, drugs and

viruses (Calderon-Garciduenas et al., 2004, 2008a,b,c;

Eriksson et al., 1999; Islam et al., 2006, 2007; Larsson

and Tjalve, 2000; Peters et al., 2006). As an example,

children and young adults exposed to severe air

pollution in Mexico City have deposits of particles in

the olfactory mucosa with transport along the olfactory

tract bulb that show up-regulation of markers of

inflammation in the frontal cortex, hippocampus, cerebellum

and alteration of the blood–brain barrier

(Calderon-Garciduenas et al., 2004, 2008a,b,c). In

addition, the translocation of ultrafine particles to the

olfactory bulbs and extra-pulmonary organs was

observed in rats (Oberdorster et al., 2004) and humans

(Peters et al., 2006) demonstrating that fine particles

target the brain and other organs, e.g. blood vessels,

cardiovascular system and kidneys. Evidence for

endothelial inflammation and damage in children was

characterized by increased TNF-a, prostaglandin E2

(PGE2), C-reactive protein and increased endotholein-

1 and down-regulation of soluble adhesion molecules

(Calderon-Garciduenas et al., 2008c). Finally, air

pollution leads to brain inflammation resembling

Alzheimer-like pathology as well as neurocognitive

deficits in young adults and children (Calderon-

Garciduenas et al., 2004, 2008a). These authors also

demonstrated fine particles within RBCs adjacent to

capillary endotheliumin the brain. In conclusion, exposure

to fine particles present in damp indoor spaces

(Gorny, 2004) that have been proven to contain

Jack D Thrasher and Sandra Crawley 591

591

trichothecenes (Brasel et al., 2005a,b; Gottschalk et al.,

2008) and other toxic metabolites (Johanning et al.,

2002a,b) must be considered as probable sources of

human neurocognitive abnormalities, described below,

Mechanism of Neurological Injury.

Mycotoxins

Mycotoxins are produced by some fungi (Campbell

et al., 2004; Jarvis, 2002; Li and Yang, 2004; Nielsen,

2003). Mycotoxin production is influenced by substrate

composition, water activity and temperature.

It is crucial to inventory indoor molds to species in

order to assess if toxigenic molds are present. Exposure

of occupants mainly results from inhalation and,

to a lesser extent, skin absorption and ingestion.

Molds produce mycotoxins during rapid growth

(Straus, personal communication). At low concentrations,

they cause mycotoxicosis in humans and animals.

The mycotoxins causing disease include

aflatoxins, ochratoxin A, trichothecenes, citreviridins,

fumonisins and gliotoxins (Bennett and Klich, 2003;

Peraica et al., 1999; Richard 2007). Mycotoxins can

regulate the immune system up or down as well as

inhibit synthesis of protein, RNA and DNA (Bok

et al., 2006; Stanzani et al., 2005). Moreover, they can

form DNA adducts (Peng et al., 2007; Pfohl-

Leszkowicz et al., 2007), protein adducts (Campbell

et al., 2004; Vojdani et al., 2003a,b; Yike et al.,

2006) and cause oxidative stress (Gardiner et al.,

2005; Peng et al., 2007) as well as mitochondrial

directed apoptosis (Chan, 2007; Stanzani et al.,

2005). Some of the animal and human health concerns

from mycotoxin-producing fungi are listed in Table 2.

Conjugation of mycotoxins to human serum albumin

and detection of the conjugates have been

reported. Aflatoxin B1-albumin adducts occur with

up to 350 pg of AFB1-lysine equivalent/mg albumin

(Wild et al., 1990). The conjugation is reported to

be permanent and irreversible (Nassar et al., 1982).

Humans form aflatoxin–albumin conjugates equivalent

to similar conjugates formed in animals sensitive

to the mycotoxin (Wild et al., 1996). Aflatoxin–albumin

adducts are present in children with impaired

growth (Gong et al., 2004) and in cases of acute aflatoxicosis

(Azziz-Baumgartner et al., 2004). Genetic

polymorphism in glutathione S-transferases affects

adducts level. Individuals with glutathione S-transferase

M1 (GSTM1) null had increased levels of adducts

versus individuals with normal GSTM1 enzymatic

activity. This enzyme conjugates aflatoxin B1 8,

9-epoxide to albumin (Chen et al., 2000; Sun et al.,

2001; Wojnoski et al., 2004). In addition, polymorphism

in CYP3A5 and CYP3A affects aflatoxin–albumin

adduct levels. CYP3A5 haplotypes with high enzyme

activity had increased levels compared to individuals

with low activity. The effect was more evident in individuals

with low CYP3A4 enzyme activity (Wojnoski

et al., 2004). More recently, attention has been directed

towards the study of S. chartarum and trichothecenes.

Albumin conjugates with satratoxin G have been

demonstrated. As many as 10 satratoxin molecules

adduct with albumin at lysyl, cysteinyl and histidyl

amino acid residues of the protein. Satratoxin

G-albumin adducts were identified in the sera of

exposed humans and rat pups (Yike et al., 2006). In

addition, antibodies to satratoxin H were present in the

systemic circulation of humans exposed to S. chartarum

(Vojdani et al., 2003a,b).

The neurotoxic mycotoxins include ergot alkaloids,

trichothecenes, citreviridin, patulin, fumonisins

and tremorgens. The neurotoxic effects of the tremorgens

in laboratory animals are on the brainstem,

stellate ganglion and Purkinje cells of the cerebellum.

The tremorgens can affect neuroreceptor sites (e.g.

gamma aminobutyric acids [GABA] and inositol 1,

4, 5-trisphosphate receptor), inhibit acetylcholinesterase,

and release excitatory neurotransmitters (e.g.

glutamate aspartate, GABA, serotonin) (Campbell

et al., 2004; Chen et al., 1999; Land et al., 1987; Selala

et al., 1989; Valdes et al., 1985). In humans, the tremorgens

verrucologen and fumitremogen C produced

by A. fumigatus have been implicated in wood trimmer’s

disease, characterized by alveolitis and tremors

(Land et al., 1987). Verruculogen decreases GABA

levels in the mouse brain (Hotujac et al., 1976).

Fumonisin-contaminated corn tortillas have been

linked to an increased risk of neural tube defects and

fetal death in residents along the Texas–Mexico

border (Missmer et al., 2006). These mycotoxins inhibit

ceramide synthase causing an accumulation of

bioactive intermediates of sphingolipid metabolism

(sphinganine and sphingoid bases). They also interfere

with folate transport and cause craniofacial defects in

mouse cultures and in utero. The administration of folic

acid or a complex sphingolipid was preventative with

respect to the in utero defects (Marasas et al., 2004).

Intrathecal instillation of extracts of P. brevicompactum

and chrysogenum that contained mycophenolic

acid and roquefortine C in mice at concentrations

of the mycotoxins ranging from 0.5 to 12.5 nM/g

body weight caused inflammation within 6 hours at

592 Toxicology and Industrial Health 25(9-10)

592

Table 2. Mycotoxins produced by toxic molds

Metabolite Disease Organisms Health Concerns

Gliotoxin Invasive aspergillosis Aspergillus fumigatus,

terres, flavus, niger,

Trichoderma virens,

Penicillium spp,

Candia albican

Immune toxicity, immune

suppression, neurotoxicity

Aflatoxin B1; kojic acid;

aspergillic acid;

nitropopionic acid

Carcinogenesis Aspergillus flavus Liver pathology and

cancer; immune toxicity;

neurotoxicity

Fumigaclavines; fumitoxins;

fumitermorgens;

verruculogen; gliotoxin

Aspergillosis Aspergillus fumigatus Lung disease; neurotoxicity;

tremors; immune toxicity

Ochratoxin A BEN Immunosuppression

Urinary tract tumors; Aspergillus niger BEN

Aspergillosis Penicillium verrucsum Lung disease

Ochratoxin A Urinary tract Aspergillus ochraceus Nephropathology

Penicillic Acid;

Xanthomegnin;

Viomellein; Vioxanthin

Tumors

Sterigmatocystin Carcinogenesis Aspergillus versicolor Liver pathology and cancer

5-methoxysterigmato cystin

Chaetomiums; Unknown Chaetomium globosum Cytotoxicity

Chaetoglobosum Cell division

A and C

Griseofulvin; Unknown Memnoniella echinata Carcinogenesis?

Dechlorogrseofulvins Reproductive toxin

Trichodermin; Hypersensitivity?

Trichodermo Protein synthesis inhibition

Mycophenolic acid Unknown Penicillium brevicompactum Cytotoxic; mutagen

Botryodiploidin Unknown Penicillium expansum Immune toxicity; cytotoxic;

Patulin; citrinin

Chaetoglobosin Tremors

Roquefortine C

Verrucosidins Unknown Penicillium plonicium Tremors, cytotoxicity;

Penicillic acid Nephropathology

Nephrotoxic glyco-peptides

Trichothecenes Unknown Trichoderma species Trichothecene toxicity

Trichodermol Immunotoxicity

Trichodermin

Gliotoxin; Viridin

Fumonisins CNS birth defects Fusarium verticillioides

(aka moniliforme)

Neural tube defects in

animals and humans

Spirocyclic Pulmonary bleeding Stachybotrys chartarum Respiratory bleeding

Drimanes; roridin Protein synthesis inhibition

Satratoxins (F, G, H) Neurotoxicity

Hydroxyroridin E Cytotoxicity

Verrucarin J Immune toxicity

Trichodermin

Dolabellanes

Altrones B, C;

Stahybotrylactams

BEN; Balkan Endemic Nephropathy

Jack D Thrasher and Sandra Crawley 593

593

concentrations of mycotoxins. Cellular and chemical

markers of inflammation were elevated, including

macrophages and neutrophils, MIP-2, TNF and IL-6

concentrations in bronchoalveolar lavage fluid

(BALF). A dose response was seen for mycophenolic

acid (macrophages) and MIP-2. In addition, brevianamide

A induced cytotoxicity with increased LDH

concentrations. Albumin, a marker of pulmonary

capillary vascular leakage, was also elevated in the

BALF (Rand et al., 2005). Finally, zearalenone and

zearalenol are estrogenic compounds already associated

or correlated with increased incidence of

infertility, abortion and uterine prolapse in livestock

(Zinedine et al., 2007). They probably have estrogenic

action in humans exhibited by precocious puberty

(Leffers et al., 2001; Massart et al., 2008).

Mycotoxins have been detected in the air and

building materials following water intrusion. Sterigmatocystin

produced by A. versicolor was detected

in 2 of 11 carpet dust samples from water-damaged

homes (Englehart et al., 2002). Bulk samples from a

Finnish building with moisture problems were analyzed

for 17 different mycotoxins. Sterigmatocystin

was present in 24% of the samples. Trichothecenes

were detected in 19% of the materials as follows:

Satratoxin G or H (five samples); diacetoxyscirpenol

(five samples); 3-acetydeoxynivalenol (three samples)

and deoxynivalenol, verrucarol or T-2 tetraol

in an additional five samples. Citrinine was found in

three samples. A. versicolor was present in most

sterigmatocystin-containing samples. Stachybotrys

spp. were present where satratoxins were detected

(Tuomi et al., 2000). Screening of dust samples from

the ventilation system of office buildings revealed the

presence of the trichothecenes, T-2 toxin, diacetoxyscirpenol,

roiridine A and T-2 tetraol (Smoragiewicz

et al., 1993). Satratoxin G and H were identified in

buildings with dampness in Denmark (Gravesen

et al., 1999) and Germany (Gottschalk et al., 2008) and

the United States (Hodgson et al., 1998). Finally,

Johanning et al. (1996, 1999, 2002a,b) demonstrated

that indoor air of S. chartarum contaminated structures

is cytotoxic in an in vitroMTT (-(4, 5-dimethylthiazolyl)-

2, 5-diphenlytetrazolium bromide) assay. MTT is a

colorimetric assay that involves the reduction by mitochondria

of living cells of the yellow MTT to purple

formazan. Trapped particulates from the indoor air of

moldy buildings contain macrocyclic trichothecenes

(satratoxins) and spirocyclic lactones. However,

mycotoxins produced by other genera of molds in the

indoor air cannot be ruled out. Thus, the MTT

cytotoxicity assay responds to mycotoxins, e.g. gliotoxin,

fumonisins, aflatoxins, patulin, etc., as well as

Type A and B trichothecenes (Hanelt et al., 1994;

Schultz et al., 2004; Smith et al., 1992; Visconti

et al., 1991; Yike et al., 1999).

The authors of this paper were involved in sampling

of three homes. Molds isolated and cultured from bulk

samples obtained from the three homes revealed

mycotoxins as follows: Home 1: satratoxins H and G,

isosatratoxin F, roridin, 1-2, E and isororidin, epoxydolabellane

A, MER 503; aflatoxin B, sterigmatocystin

and cyclopanzoic acid; Home 2: roquefortine C, sterigmatocystin

and 5-methyloxysterigmatocystin; and

Home 3: sterigmatocystin, MER 503 and dolabellanes

(Neville, P-K Jarvis unpublished reports). An

18-month-old male child in one of the homes died from

pulmonary bleeding. In the other two homes, two

women and a 7-year-old boy developed permanent

neurocognitive deficits as well as increased sensitivity

to various odorous chemicals. The latter three had

changes in quantitative electroencephalograms

(QEEG) involving the frontal cortex as well as other

regions of their brains. The neurocognitive deficits

were shown by testing performed by neuropsychologist

Raymond Singer, PhD Santa Fe, New Mexico.

Airborne macrocyclic trichothecenes in contaminated

buildings, control buildings and outdoor air

were investigated (Brasel et al., 2005a,b). The Quant

Tox Kit manufactured by Envirologix was utilized to

detect satratoxin G and H, verrucarin A, verracarol

and isosatratoxin F by an ELISA method with roridin

A as the control. Air samples were collected using a

Spin Con PAS bioaerosol sample. The air samples

were pulled through multistage polycarbonate filters

of 5.0, 1.2 and 0.4 mm. The mycotoxins were present

in all particulate fractions, particularly 0.4 to 1.2 mm.

Briefly, macrocyclic trichothecene concentrations

present in the fine particle fractions ranged from

<10 to >1300 pg/m3, significantly greater (p < .001)

than detection in control buildings and outdoor air.

In addition, the trichothecenes were detected in the

sera of symptomatic occupants of the same buildings

vs controls (p < .05; Brasel et al., 2004). More

recently, elevated macrocyclic trichothecenes were

reported in flooded moldy dwellings in which S. chartarum

was present (Charpin-Kadouch et al., 2006).

Additionally, Bloom et al. (2007), using gas chromatography

as well as HPL with tandem mass spectrometry,

tested for the presence of trichothecenes

(verrucarol, trichodermol, satratoxins G and H, trichodermol,

gliotoxin, aflatoxins and sterigmatocystin) in

594 Toxicology and Industrial Health 25(9-10)

594

building materials and dust from water-damaged

buildings and homes. Of 62 samples, 45 were positive

for mycotoxins, three of eight settled dust samples

and five of eight air dust samples were positive for

macrocyclic trichothecenes and sterigmatocystin.

Additionally, concentrations of various mycotoxins

were as follows: building materials (gliotoxin at

0.43-1.12 pg/mg; sterigmatocystin at 4.9-50,000 pg/

mg; trichodermol at 0.9-8700 pg/mg; verrucarol at

8.8-17,000 pg/mg and dust samples (aflatoxin B1 at

32.0-13,500 pg/cm2; sterigmatocystin at 3.6-

10,900 pg/cm2; trichodermol at 6.5-170 pg/cm2; verrucarol

at 25-3,400 pg/cm2; gliotoxin at 400 pg/cm2).

In addition, verrucarol and sterigmatocystin were

found in dust samples from Katrina homes (Bloom,

2008). Also, airborne satratoxin G and H were

demonstrated in a contaminated home utilizing a

0.8-mm filter and LC-MS/MS (Gottschalk et al.,

2008). More recently, hydrophilic fungi and ergosterol

were shown to be associated with respiratory illness

in a water-damaged building (Park et al., 2008).

Ergosterol is a biomarker for the assessment of mold

damage (Foto et al., 2005; Hippelein and Rugamer,

2004). In conclusion, mycotoxins in damp indoor

environments become airborne in both large (spores,

hyphae fragments) and fine particles. They are also

present in bulk in dust samples from the same

buildings. In conclusion, multiple mycotoxins, e.g.

trichothecenes, aflatoxins, gliotoxin, are prevalent in

water-damaged homes and buildings.

The epipolythiodioxopiperzines (ETP) are a class of

fungal toxins produced by several different genera of

mold (Gardiner et al., 2005). One of the most abundant

ETP is gliotoxin produced by A. fumigatus, niger, terreus,

flavus, Trichoderma virens, Penicillium spp. and

C. albicans (Gardiner et al., 2005; Lewis et al., 2005b).

Gliotoxin is a virulence factor in invasive A. fumigatus

in mice and probably for humans (Kupfahl et al., 2008;

Lewis et al., 2005a,b; Sugui et al., 2007). Gliotoxin is

an immunomodulating toxin with suppressive activity

(Mullbacher et al., 1986; Sutton et al., 1994). It inhibits

macrophage and polymorphonuclear cell function and

generation of alloreactive cytotoxic T cell. The toxin

inhibits the transcription factor, nuclear factor kappalight-

chain-enhancer of activated B cells (NF-kB), an

integral part of the inflammatory immune response and

controls expression of some cytokines. Finally, gliotoxin

and other ETPs are mitochondrial poisons resulting

in reduction of adenosine triphosphate (ATP) by

hyper-polarization of the mitochondrial membrane and

causing apoptosis (Gardiner et al., 2005; Pardo et al.,

2006). Gliotoxin has been identified in the lungs and

sera of mice and cancer patients with aspergillosis

(Lewis et al., 2005a). The percentage of Aspergillus

species isolated from cancer patients with IA secrete

gliotoxins as follows: A. fumigatus – 93%; A. niger

75%; A. terres – 24%; and S. flavus – 4% (Lewis

et al., 2005b). Moreover, the production of gliotoxin

by clinical and environmental isolates of A. fumigatus

has been confirmed in Germany and Austria (Kupfahl

et al., 2008). The percentage of A. fumigatus isolates

that produced gliotoxin was clinical isolates – 98%;

environmental isolates – 96%. The toxin was also

detected in decreasing frequency in other isolated species:

A. niger – 56%; A. terreus – 37%; and A. flavus

13%. In conclusion, these observations make it

imperative that more attention should be paid to

Aspergillus species as well as other genera of molds

and their production of gliotoxin. The need for an

increased awareness of these molds is apparent with

respect to the exposure of humans who have risk

factors of corticosteroid usage, COPD, diabetes mellitus,

pre-existing illnesses as well as altered immune

function, e.g. autoimmune diseases.

Mechanism of neurological injury

Three independent sets of information have been used

to discuss a plausible mechanism for neurological

impairment observed in humans exposed to contaminated

air. The first set includes clinical observations

on humans exposed to water-damaged environments.

The second entails animal experiments demonstrating

neurological injury from mycotoxins instilled into the

olfactory mucosa. The third set of data involves clinical

and pathology of brain injury to children and

young adults exposed to the polluted air of Mexico

City.

Clinical findings in patients exposed to waterdamaged

buildings: Both central and peripheral neuropathy

have been reported in individuals chronically

exposed to damp indoor environments (Campbell

et al., 2003; 2004; Crago et al., 2003; Gordon and

Cantor, 2004, 2006; Gray et al., 2003; Kilburn,

2003, 2004; Rea et al., 2003). Briefly, exposed

individuals develop peripheral neuropathy with

autoantibodies directed against several neural antigens

(Campbell et al., 2004). Toxic encephalopathy

involves multiple symptoms, including loss of

balance, recent memory decline, headaches, lightheadedness,

spaciness/disorientation, insomnia, loss

of coordination (Gray et al., 2003; Kilburn 2003,

Jack D Thrasher and Sandra Crawley 595

595

2004; Rea et al., 2003). Exposed individuals had

alterations in QEEG involving the frontal cortex and

other regions of the brains (Crago et al., 2003)

coupled with neurocognitive decline (Crago et al.,

2003; Gordon and Cantor, 2004, 2006; Kilburn,

2003, 2004) as well as significant changes in various

neurological measurements (declines in simple reaction

and choice reaction times, increased body sway

with eyes open and closed, increased latency of blink

reflex, and decreased grip strength, among others

(Kilburn, 2003, 2004). The probable explanation of

the causative mechanism comes from both animal

models and humans exposed to air pollution.

Instillation of mycotoxins into the olfactory mucosa

of rodents: Satratoxin G, roridin A and aflatoxin B1

instilled into the olfactory area cause sensory olfactory

neuron loss, nasal and brain inflammation and

neurotoxicity. The mycotoxins are transported into

the brain along the olfactory tract leading to inflammation

and damage in the tract and the olfactory

bulbs. Tritium labelled aflatoxin B1 at 0.2, 1 or

20 mg was intranasally instilled in rats and followed

by autoradiography and spectrometry. The mycotoxin

was bioactivated in the olfactory/nasal mucosa and

transported along the olfactory tract to the bulbs.

Twenty-four hours after instillation, the olfactory

epithelium was disorganized and undulating with

pyknotic nuclei, shrunken cytoplasm and transport

of the labelled aflatoxin to the olfactory bulbs. The

pathology was still present at 5 days post instillation

at 20 mg (Larsson and Tjalve, 2000). Satratoxin G was

instilled into the olfactory mucosa in mice at 5 and

25 mg/kg body weight. Apoptosis of olfactory neurons

occurred along with the release of proinflammatory

cytokines TNF-a, IL-6, IL-1 and MIP-2 in the nasal

airways, ethmoid turbinates and olfactory bulbs.

Marked atrophy of the olfactory nerve and glomerular

layers of the bulb were observed (Islam and Pestka,

2006; Islam et al., 2006). Similarly, roridin A instilled

into the olfactory mucosa of mice at 500 mg/kg body

weight induced apoptosis of olfactory neurons, atrophy

of the olfactory epithelium and olfactory bulbs.

The kinetics of the reported pathology was

potentiated by the simultaneous exposure to lipopolysaccharide

(Islam et al., 2007). Also, lipopolysaccharides

enhance the hepatoxicity of aflatoxin B1 in rats

(Barton et al., 2001; Luyendyk et al., 2002, 2003).

Finally, C-14 aromatic carboxylic acids are transferred

unchanged into the brain and olfactory bulbs

following intranasal instillation in mice (Eriksson

et al., 1999). These observations point towards at least

one probable mechanism for the encephalopathy

observed in humans exposed to the biocontaminants

in damp indoor spaces.

Children and young adults living in Mexico City:

Additional evidence for the role of particles and

associated toxins as causation in the onset of toxic

encephalopathy is derived from observations of

children and young adults residing in Mexico City.

Particulate matter in polluted outdoor air consists

of fine and ultrafine particles to which toxins are

adsorbed.

Autopsies were performed on children and young

adults who had died suddenly and who did not have

familial or personal history of neurological disease.

Inhaled particles were observed by electron and light

microscopy. They were distributed to organs (liver,

spleen, kidneys, brain, within RBCs and heart), via

the systemic circulation and/or by macrophage and

dendritic cell activity and via the olfactory mucosa.

Observations on the brains showed marked upregulation

of inflammatory markers in the frontal cortex,

olfactory bulb, substantia nigrae, vagus nerve and

disruption of the blood–brain barrier. Pathological

observations included deposition of ultrafine particles,

accumulation of amyloid b-42, a synuclein and

increased expression of COX2 in the brains. These

findings were observed in the frontal cortex, olfactory

bulb, substantia nigrae and vagus nerve of affected

individuals (Calderon-Garciduenas et al., 2004,

2008a,b,c; Peters et al., 2006). The pathology

resembled that of Alzheimer- and Parkinson-like

diseases (Calderon-Garciduenas et al., 2004; 2008b).

Clinically healthy children exposed to air pollutants

have systemic inflammation and endothelial

damage with significant increases in inflammatory

markers (TNF-a, PGE2, C-reactive protein, IL-1b,

endothelin-1) with a concomitant down-regulation of

soluble adhesion molecules (Calderon-Garciduenas

et al., 2008c). Finally, exposed children exhibited significant

deficits in short- and long-term cognition with

neuropsychological testing. Over 50% of them had

magnetic resonance imaging (MRI) findings of prefrontal

white matter hyperintense lesions. Concomitantly

exposed canines had the same MRI changes

and increases in COX2 inflammatory markers, i.e. neuroinflammation

(Calderon-Garciduenas et al., 2008a).

In conclusion, published observations point towards

the role of fine particles in the exposure of occupants in

water-damaged structures to mycotoxins and other biocontaminants

(see below Endotoxins and MVOCs/

VOCs). The observations include (1) particulates

596 Toxicology and Industrial Health 25(9-10)

596

<1.5 mm contain trichothecenes and are associated with

growth of the mold (Brasel et al., 2005a,b); (2) the trichothecenes

were detected in the sera of symptomatic

individuals occupying the S. chartarum contaminated

structures (Brasel et al., 2004); (3) trichothecenes and

other mold by-products are present in particles

<2 mm in other settings and are cytotoxic in the MTT

assay (Johanning et al., 2002a,b); (4) trichothecenes are

in the urine, blood, nasal and lung secretions of individuals

exposed to molds in water-damaged homes (Croft

et al., 1986; 2002; Hooper, 2008, personal communication);

(5) finally, the blood concentrations of the

haemolytic protein (stachylysin) of five symptomatic

individuals exposed to S. chartarum averaged

371 nanograms/mL (Van Emon et al., 2003) and (6)

toxic strains of Bacilli, Nocardia and Streptomyces

were isolated from indoor air in the particle range of

0.56 to 2.1 mm (Peltola et al., 2001a,b).

VOCs and MVOCs

In general, VOCs and MVOCs are present in the

indoor environment. Some of the sources for VOCs

are building materials, cleaning agents, personal care

products (perfumes), paints, furnishings, microbial

growth, etc. (Kim et al., 2007; Lee et al., 2005). In

addition, fungi and bacteria also produce VOCs, usually

referred to as microbial MVOCs. The composition

MVOCs varies according to substrate, humidity

and species (Claeson and Sunesson, 2005; Gao and

Martin, 2002; Gao et al., 2002; Korpi et al., 1999; Nilsson

et al., 2004; Sunesson et al., 1996). The emitted

MVOCs include limonene, hexanol, acetone, butanone,

pentanone, 2-ethyl-1-1 hexanol, 1-butanol, 3-methyl-1-

butanol, 2-methyl-1-propanol, terpineol, 2-heptanone,

1-octen-3-0l, dimethyl disulfide, 2-hexanone,

3-octanone, 2 pentylfuran, aldehydes, ammonia and various

amine compounds (Claeson and Sunesson, 2005;

Gao and Martin, 2002; Gao et al., 2002; Li and Yang,

2004; Nilsson et al., 2004; Korpi et al., 1999; Sunesson

et al., 1996). For the sake of further discussion, the

MVOCs and VOCs will be considered as VOCs.

Porous materials act as a sponge-adsorbing VOC,

the latter from which re-emission occurs. Thus,

regardless of the origin of the VOCs, adsorption to

indoor dust, particles and porous surfaces occurs.

Inhalation of dust and particles leads to deposition

of the VOCs in the olfactory mucosa as well as the

respiratory tract (Gorny, 2004; Nilsson et al., 2004;

also see above fine particulates). Indoor VOC concentrations

are higher than outdoor concentrations,

increasing human exposures to toxins (Kinney et al.,

2002; Wallace et al., 1991). Children living in dwellings

with elevated VOCs from microbes have a higher

prevalence of asthma, fever, wheezing and irritation

of the eyes (Elke et al., 1999). Fungal-related VOCs

in damp buildings have been associated with

increased nasal biomarkers of inflammation (cationic

proteins, myeloperoxidase and albumin), increased

blinking and a decrease in forced vital volume (FVC)

(Walinder et al., 2001, 2005). In addition, fungal colonization

of fiberglass insulation leads to the distribution

of VOCs through the air conditioning system,

which may be related to sick building syndrome

(Ahearn et al., 1996, 2004). In conclusion, more attention

needs to be paid to the contributions of VOCs to

the adverse health effects in individuals residing in

water-damaged building.

Extracellular proteins, enzymes, siderophores

and haemolysins and pulmonary haemorrhage

Molds excrete extracellular enzymes and proteins to

digest and absorb nutrients from substrates that

include lipases, proteinases, chitinases, amylases,

esterases, phospho-lipases, siderophores and haemolysins,

among others (Birch et al., 2004; Donohue

et al., 2005, 2006; Hu et al., 2004; Kudanga et al.,

2007; Mellon et al., 2007; Moon, et al., 2006; Schretti

et al., 2007; Vesper and Vesper, 2004; Vesper et al.,

2004; Yike et al., 2007). Inhaled microbial proteinases

cause inflammation in the respiratory tract,

activating protease receptors with production of

IL-6, IL-8 and release of IL6, IL-8, PGE2,

granulocyte-macrophage colony-stimulating factor

(GMCSF). Neutrophils and eosinophils are recruited

(Asokananthan et al., 2002; Chiu et al., 2007; Reed,

2007; Shin et al., 2006; Yike et al., 2005, 2007). In

addition, siderophores that bind iron have a distinct

role in A. fumigatus infections (Schretti et al., 2007).

An outbreak of infantile pulmonary haemosiderosis

in Cleveland was associated with S. chartarum. A haemolysin

(stachylysin) and a siderophore were identified

from strains of Stachybotrys isolated from the infants’

homes and from a lung of a child with pulmonary

haemorrhage (Dearborn et al., 2002; Elidemer et al.,

1999; Vesper et al., 2000). The Cleveland cases were

criticized by the CDC for statistical errors and limitations

in sampling procedures during the initial evaluation

of the affected homes (CDC, 2000). However,

recent observations indicate that species of mold other

than S. chartarum secrete haemolysins.

Jack D Thrasher and Sandra Crawley 597

597

Several of the mold genera were isolated from the

dust of the Cleveland case homes. They were

identified to species by quantitative polymerase chain

reaction. The isolates were tested for the production

of haemolysins (Vesper and Vesper, 2004). Eleven

species of Aspergillus, ten species of Penicillium, two

species of Ulocladium, Paecilomyces variotil, Memnoniella

echinata, Scopulariopsis brevicaulis, Trichoderma

longibrachiatum and viride and S. chartarum

were demonstrated to cause haemolysis of sheep’s

blood agar. Haemolysins were more often produced

by the fungi from homes with pulmonary haemorrhage

(42%) than from reference homes (10%). These

observations emphasize the complexity of damp

indoor spaces and broaden the possible biological

agents responsible for the adverse health effects to

occupants of water-damaged indoor environments.

Galactomannans (EPS)

Galactomannans are cell wall polysaccharides

consisting of a mannose back bone with galactose side

groups. Other sugars include glucose, rhamnose, arabinose

and xylose. They are highly branched with 1-2,

1-5 and 1-6 linkages and are released from the cell

wall during growth (Notermans and Soentoro, 1986;

Notermans et al., 1987, 1988). EPS are antigenic with

1-5 linked bD-galactofuranosides being immunodominant

from Aspergillus/Penicillium species (Kamphuis

et al., 1992; Notermans et al., 1988). Antibodies

against EPS have been detected in the sera of animals

and humans (Notermans et al., 1987, 1988). EPS are

present in the sera of immunocompromised organ

transplant patients with IA (Pfeiffer et al., 2006). In

addition, antigenic EPS are produced by species of

Rhizopus, Mucor, Rhizomucor, Absidia cormybifera

and Syncephalastrum racemosum (De Ruiter et al.,

1992). EPS are readily detected in house dust of homes

with reported dampness and are associated with

respiratory symptoms in children (Douwes et al.,

1999, 2003, 2006). They and 1-3-bD-glucans are good

markers for the overall level of fungal concentrations

in dust and as a surrogate for estimating airborne fungal

exposure (Chew et al., 2001). Thus, EPS are additional

biocontaminants in damp indoor spaces and appear to

be associated with respiratory symptoms in children.

1, 3-bD-glucans

The 1, 3 bD-glucans (glucans) are diagnostic markers

for fungal infections, particularly, Aspergillus species,

systemic candidiasis, and other fungi (Kondori

et al., 2004; Pazos et al., 2005; Pickering et al.,

2004). However, PCR DNA analytical tests give earlier

and more specific diagnostic results for infections

(Khan et al., 2007; Musher et al., 2004; Rantakokko-

Jalava et al., 2003).

The glucans have been demonstrated in the indoor

air and dust and their presence is related to fungal

growth and possible intrusion from outdoor sources

(Chew et al., 2001; Douwes et al., 2006; Gehring

et al., 2001; Rylander, 1999, 2004). The glucans cause

airway inflammation. They have been identified in

bronchoalveolar lavage fluid from individuals with

acute eosinophilic pneumonia (Kawayama et al.,

2003; Thorn and Rylander, 1998). Inhalation of glucans

by healthy individuals caused an increase in

eosinophilic cationic protein, TNF-a and a reduction

of peripheral blood eosinophil numbers (Thorn et al.,

2001). Similar observations have been reported in guinea

pigs and mice treated with glucans (Fogelmark

et al., 2002). In contrast, blood leukocytes from

healthy volunteers and patients allergic to house dust

glucans enhance the release of IgE and histamine in

vitro (Holck et al., 2007). Thus, the effects of inhaled

glucans may be different in eosinophilic pneumonia

cases vs healthy and allergic individuals when tested

in vivo compared to in vitro assays. Airway inflammation

in adults with chronic exposure to glucans is

associated with increased prevalence of atopy, a slight

increase in myeloperoxidase and a decrease in forced

expiratory volume (FEV1; Thorn and Rylander,

1998). Moreover, children exposed to glucans in dust

at home and at school have variability in pulmonary

peak flow values as well as signs of airway inflammation

(Douwes et al., 2000; Rylander, 1997, 1999;

Rylander et al., 1998) and have a higher incidence

of infections (Rylander, 2004). Finally, nasal deposition

of glucans is not associated with acute

inflammation with respect to an increase of the

chemo-attractant eotaxn and eosinophils in nasal

lavage fluid (Beijer and Rylander, 2005). Inflammatory

response to 1-3 bD-glucans involves toll-like

receptors 2 (TLR2) and TLR4 receptors, MYD88 and

Dectin-1 (Hohl et al., 2005; Meier et al., 2003; Wang

et al., 2002). Finally, antibodies against glucans have

been demonstrated in humans and animals exposed to

molds (Kamphuis et al., 1992; Notermans et al., 1987,

1988). The detection of glucan antibodies suggests

that inhalation of glucans and/or colonization/infection

has occurred. In either event the antibodies to

glucans demonstrate an immune response unrelated

to IgE sensitivity. Also, glucans are in the blood of

598 Toxicology and Industrial Health 25(9-10)

598

patients with deep invasive mycosis and fungal febrile

episodes and can be used to diagnose infections

(Kedzierska, 2007; Miyazaki et al., 1995; Obayashi

et al., 1995; Pickering et al., 2004). Additional work

is needed to determine the role of glucans in respiratory

inflammation. However, children seem to be

more susceptible to exposure.

Endotoxins

Endotoxins are LPS complexes of the outer cell wall

of gram-negative bacteria, usually pathogens such as

E. coli, Salmonella, Shigella and Pseudomonas, etc.

The LPS are maintained within the outer cell wall

until autolysis of the bacteria, which releases them

into the surrounding environment. They are pyrogenic

(fever producing), antigenic and cause inflammation

through the activation of the complement system via

CD14 protein, the TLR4-signaling pathway and

release of inflammatory cytokines, e.g. TNF-a.

CD14 protein binds LPS and transfers them to the

TLR4 receptor. Clinical or experimental outcomes

include fever, leukopenia, hypoglycemia, hypotension,

impaired perfusion of essential organs (brain,

heart, kidney), activation of C3 and the complement

cascade, bleeding, intravascular coagulation, septic

shock and death. In addition, LPS also cause an

increased production of the long pentraxin PTX3

(Cunningham et al., 2005; Imamura et al., 2007) and

in the maturation of dendritic cells evoking Th1 and

Th17 responses (Iwamoto et al., 2007). LPS are present

in the indoor environment of normal and waterdamaged

homes and buildings (Douwes et al., 2006;

Gorny, 2004; Gorny et al., 2002; Park et al., 2000,

2006; Rao et al., 2007b).

In transgenic mouse models, endotoxins interact

with the TLR4-signaling pathway, CD14 phenotype,

TNF-a and other factors leading to increased airway

inflammation (Jung et al., 2006; Martinez, 2007a,b;

Togbe et al., 2007). In addition, in vitro and in vivo

animal models of neurological diseases have shown

that intra-peritoneal (i.p.), i.v. and intracerebral

administration cause expression of proinflammatory

markers of microglia (Qin et al., 2004) as well as the

induction of oligodendrocyte injury via TLR4

(Lehnardt et al., 2002). Intracerebral or systemic

administration of endotoxin exacerbates microglial

inflammatory response and increases neuronal cell

death in ME7 prion mouse model (Cunningham

et al., 2005). Moreover, systemic inflammation (e.g.

infectious states) appears to be involved in chronic

neurodegenerative disease (e.g. Alzheimer, Parkinson).

The increased synthesis of inflammatory cytokines

and other mediators during infections and/or

systemic LPS challenge promote an inflammatory

response that may contribute to the progression of

chronic neurological disease (Cunningham et al.,

2005: Godbout et al., 2005; Perry, 2004; Polentarutti

et al., 2000). Co-exposure of mice to vomitoxin and

LPS caused a synergistic increase in TNF-a messenger

RNA (mRNA) as well as plasma TNF-a and

IL-6. Marked cell death (apoptosis) and loss occurred

in the lymphatic organs, thymus, Peyer’s patches,

spleen and bone marrow (Islam et al., 2002; Zhou

et al., 1999, 2000). The priming of mice with LPS

lowered the dosage of deoxynivalenol causing upregulation

of inflammatory cytokines (IL-a and –b,

IL6 and TNF-a) and massively increased the thymus

apoptosis (Islam et al., 2002). Similarly, in vitro priming

of TLR of murine macrophages and human whole

blood cultures renders macrophages sensitive to

exposure to mycotoxins and other xenobiotics. The

LPS-sensitized macrophages have an increased

production of mRNA of IL-1b, IL-6 and TNF-a after

exposure to deoxynivalenol (DON), satratoxin G and

zeralenone (Pestka and Zhou, 2006). Also, administration

of aflatoxin B1 and endotoxin to rats augments

liver sinusoidal damage and clotting by converting

soluble fibrinogen to insoluble fibrin clots (Luyendyk

et al., 2003). Finally, nasal inflammation, inflammatory

cytokine production and atrophy of the olfactory

nerve and olfactory bulbs in mice are enhanced by the

co-administration of LPS and roridin A (Islam et al.,

2007). In conclusion, mold agents and LPS exposure

are synergistic with adverse effects on organ systems,

including the brain, leading to a systemic inflammatory

response.

Inhaled LPS causes adverse airway responses in

healthy individuals as well as individuals with asthma

and other respiratory conditions. Healthy volunteers

challenged with LPS had variable airway responsiveness

(Kline et al., 1999). Eight sensitive subjects had

at least a 20% decline in the FEV1, at a dose of 6.5 mg

or less, while 11 hyporesponsive subjects maintained

an FEV1 at least 90% after inhaling 41 mg of LPS.

Peripheral monocytes from the hyporesponsive

individuals released fewer IL-6 and IL-8 than the

sensitive subjects.

The interaction between the environment and lung

responsiveness is a complicated gene-environment

interaction (Martinez, 2007a). The interactions

involve TLR2 and -4 and IL-1 receptors as well as

Jack D Thrasher and Sandra Crawley 599

599

polymorphism of CD14 protein (Liebers et al., 2008;

Martinez, 2007b; Simpson et al., 2006). In addition,

down-stream adaptor molecules, e.g. My88 and

TRAM, are also involved (Tanimura et al., 2008).

Furthermore, other genes (e.g, IL-13, DEFB1, TLR2,

TRL4) seem to have a role in the phenotypic complex

condition referred to as asthma. Apparently, IgEmediated

conditions are not the norm, while the role

of IL-4, IL-5, eosinophils, and neutrophils are difficult

to control (Martinez, 2007a,b). Thus, children

with CC genotype at –159 of CD14 have a decreased

risk of allergic sensitization to endotoxins while

having an increased risk of non-atopic wheezing

(Simpson et al., 2006). Also, it has been shown that

the CC allele of CD14 is a risk factor for allergic phenotypes

at a low concentration of endotoxins, whereas

the TT allele is a risk factor for higher concentrations

of LPS (Martinez, 2007b). In conclusion, gene and air

pollution interactions in asthma and endotoxins are

complex and require more genome-associated studies

with better assessment of exposure and phenotype

(London, 2007).

In conclusion, synergism between endotoxins and

mycotoxins has been demonstrated in vitro and in animal

models. As discussed above, LPS enhance the

damage to the olfactory epithelium, tract and bulb

of roridin A in mice. In addition, exposure to LPS and

aflatoxin B1 enhances liver toxicity in rats. Treated

animals had damage to sinusoidal cells and hepatocytes

with increased alanine aminotranserase and

fibrin deposition (Barton et al., 2001; Luyendyk

et al., 2002, 2003). Oral administration of vomitoxin

with simultaneously injected LPS in mice produced

a significant enhancement of TNF-a, IL-6 and IL-1b

in spleen cells (Zhou et al., 1999). A similarly

designed study resulted in an increase of apoptosis

of lymphocytes in the spleen, thymus and Peyer’s

patches (Zhou et al., 2000). Finally, in vitro priming

of murine and human whole blood macrophages

enhances the proinflammatory cytokine production

(Pestka and Zhou, 2006). Two questions arise from

these observations: (1) What role does the genetic

polymorphism CD14 protein have in synergism of

LPS and mycotoxins? (2) Are the children with

CD14 CC genotypes more or less sensitive to the

inflammatory conditions caused by mycotoxins?

Discussion and Conclusion

Damp indoor spaces create environments in which

molds and gram negative and positive bacteria

flourish. In the process of normal organism growth

or in response to a change in environmental conditions

(e.g. less moisture, human activity, etc.), the

microorganisms produce a variety of biocontaminants

that impinge upon occupants. A comprehensive

review of published peer-reviewed literature, as we

have done, clearly shows evidence of deleterious

effects on occupants exposed to indoor biocontaminants.

Data from in vitro and animal models support

this conclusion. The weight of the findings lies with

multiple authors publishing in a variety of professional

journals, who have arrived at similar conclusions.

For example, asthma in adults and children as

well as clusters of rheumatic conditions have been

attributed to fungal and bacterial contaminants (Jaakkola

and Jaakkola, 2004; Luosojarvi et al., 2004;

Myllykangas-Luosujarvi et al., 2002; Nevalainen and

Seuri, 2005; Park et al., 2008). Neurological sequelae

have been reported to include peripheral and central

neuropathy, alterations in QEEG and neurocognitive

deficits (Campbell et al., 2003, 2004; Crago et al.,

2003; Gordon and Cantor, 2004, 2006; Kilburn,

2003, 2004; Rea et al., 2003). In addition, multiorgan

symptoms are present in occupants (Croft

et al., 1986, 2002; Gray et al., 2003; Hodgson et al.,

1998; Johanning et al., 1996, 1999; Rea et al.,

2003). Finally, trichothecenes and stachylysin were

demonstrated in the sera of individuals exposed to

S. chartarum (Brasel et al., 2004; Van Emon et al.,

2003). These observations and others cited in this

review indicate that a systemic inflammatory

response is occurring and the synergism of the biocontaminants,

particularly LPS and mycotoxins,

probably plays a significant role in this response.

Regardless of the sizeable body of evidence documented

in scientific and medical literature, the major

focus of governmental agencies and medical universities

has been directed towards allergies and asthma in

regard to mold exposures. However, the literature

indicates otherwise. Occupants exposed to multiple

biocontaminants in indoor environments develop

multi-organ symptoms indicating that a systemic

inflammatory response is occurring. A general systemic

inflammatory is characterized by the presence

of proinflammatory agents (TNF-a, IL-1, myeloperoxidase,

C-reactive proteins, neutrophils, lymphocyte

activation markers, etc.) in the systemic circulation

(Rylander, 2004). Furthermore, when the synergism

and interactions of the biocontaminants are considered,

it can only be concluded that multiple systemic

health effects in humans and animals are not only

600 Toxicology and Industrial Health 25(9-10)

600

occurring but are scientifically and medically explicable.

For example, the toxic interactions between LPS

and mycotoxins are synergistic in vitro and in vivo.

Additional examples include cytotoxicity of spores

from co-cultured S. chartarum and S. californicus. The

toxic effects mimic chemotherapeutics doxorubicin

and actinomycin D. The gram positive Actinomycetes

(Streptomyces and Nocardia) and other gram positive

bacteria produce exotoxins that damage mitochondria.

Streptomyces, Nocardia and Mycobacterium species

are potential human pathogens with corticosteroids,

diabetes mellitus, COPD and immunecompromised

conditions as risk factors. Streptomyces and Mycobacterium

spp. cause hypersensitivity pneumonitis. Of

concern is the rise of biocontaminant-related diseases

worldwide, which must be paid heed. According to the

American Thoracic Society, NTM is increasing in

immunocompetent individuals. Since Mycobacterium

is a common environmental contaminant, attention

should be paid to both indoor and outdoor sources.

In addition, IA is also on the increase in immunocompetent

patients. The risk factors for IA are corticosteroids,

diabetes mellitus, COPD and extensive use of

antibiotics. It is important to understand that corticosteroids

inhibit the oxidative pathways of AM, which

are the first line of innate immune system defense

against foreign organisms. Finally, gliotoxin, a virulence

factor, is produced by both clinical and environmental

isolates of A. fumigatus, A. terreus, A. niger

and A. flavus. Furthermore, gliotoxin has been identified

in the sera and lung secretions in humans and

mice with aspergillosis. We must determine the significance

of gliotoxin in water-damaged buildings and

its correlation with the rise of IA in immunocompetent

individuals.

Currently, the concept of Th1/Th2 interactions in

inflammatory response to molds is under challenge.

Apparently, mouse and human Th17 cells, IL-17,

IL-23 and related mediators have a key role in promoting

inflammation and impair antifungal immune

resistance in lungs and CRS (Romagnani, 2008;

Romani and Puccetti, 2008; Romani et al., 2008;

Zelante et al., 2007). Interestingly, tryptophan

catabolites (kynurenines) and mouseTreg T cells have

a protective effect in taming overzealous exaggerated

inflammatory responses. IL-17 and IL-23 pathway

down regulates the tryptophan catabolism (Belladonna

et al., 2006; Romani et al., 2008). Other

advances in understanding the inflammatory response

to bacteria and molds are occurring rapidly. Long

PTX3 (prototype protein of pentraxin) is essential for

resistance to A. fumigatus and other pathogens

(Gaziano et al., 2004). PTX3 is released from lung

epithelial cells, dendritic cells, macrophages and neutrophils

by TNF-a, IL-10 and endotoxins (Doni et al.,

2006; Imamura et al., 2007; Jaillon et al., 2007).

Increased expression of PTX3 leads to an enhancement

of acute lung injury and inflammation (Gaziano

et al., 2004; Han et al., 2005). On the other hand, in a

mouse model of aspergillosis, administration of

appropriate doses of PTX3 gave complete resistance

to infection and reinfection. The protective effect was

similar or superior to that observed with liposomal

amphotericin B or deoxycholate amphotericin B. In

addition, PTX3 accelerated recovery of phagocytosis

and Th1 lymphocytes with a concomitant decrease in

inflammation. Interestingly, PTX potentiated the therapeutic

efficacy of sub-optimal doses of deoxycholate

and amphotericin B (Gaziano et al., 2004). Thus,

recent advances in research have revealed that reactive

proteins (PTX3) and Th17 lymphocytes impart

pathology observed in chronic inflammatory conditions

in humans and rodents. The recent information

clearly demonstrates that non-IgE allergic mechanisms

have a major role in chronic inflammation

caused by microbial infections (Chenz and O’Shea,

2008; Gaziano et al., 2004; He et al., 2007; Iwamoto

et al., 2007; Korn et al., 2007; Mauri et al., 2008; Muller

et al., 2001; Romagnani, 2008). At least two of the

mediating factors are long PTX3 and IL-17. It appears

from the reviewed literature that long PTX3 is the

inflammatory reactive protein that should be monitored

in a variety of chronic diseases.

In conclusion, the medical profession worldwide

should add to its basic curriculum detailed information

on the health effects of the multi-biocontaminants present

in water-damaged buildings. Diagnostic tests

should be developed and recommended to determine

the nature of building-related illness, e.g. allergy,

hypersensitivity pneumonitis, encephalopathy, fungal

infections, bacterial infection, etc. Finally, the medical

profession must recognize the importance of immediate

removal of occupants from the toxic environment.

Government agencies and medical universities need

to increase research to continue to further solidify

knowledge regarding health impacts that multibiocontaminants

have on human and animal occupants.

Preventing exposure to indoor biocontaminants is the

most effective way for society to avoid the illnesses

they cause. When exposure has already occurred,

immediate removal of the occupant(s) from the

contaminated environment is paramount and will

Jack D Thrasher and Sandra Crawley 601

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minimize further damage to health. Proper diagnoses

will enable affected individuals to either remediate the

contaminated structures, if possible, or locate other

housing and/or work environments. Increased awareness

of the potential health hazards of indoor biocontaminants

is the first step in managing – and ultimately

reducing – the illnesses they induce. As pointed out

in the preface to this issue, ‘‘If everything has to be

double-blinded, randomized and evidence-based,

where does that leave new ideas?’’