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Global Indoor Health Network 

Discussion of Mycotoxins

Discussion of 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 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).
Mycotoxins are found in water-damaged buildings
Molds and mycotoxins are found in water-damaged buildings

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 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.

Source: Thrasher, JD, Crawley SL (2009). The Biocontaminants and Complexity of Damp Indoor Spaces: More than What Meets the Eyes. Toxicology and Industrial Health. 25(9-10), 583-615. 
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